Impaired function of rDNA transcription initiation machinery leads to derepression of ribosomal genes with insertions of R2 retrotransposon

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In D. melanogaster, rDNA clusters on the X and Y chromosomes usually consist of 200-250 rDNA units, more than half of which contain insertions.

“In D. melanogaster, rDNA clusters located in the X and Y chromosomes usually consist of 200–250 rDNA units (32), more than half of which contain insertions.”

• cites [32] Ritossa, 1966, A molecular explanation of the bobbed mutants of drosophila as partial deficiencies of “ribosomal” DNA, Genetics sourceDisputed

  The citation is attached to the claim that X- and Y-chromosome rDNA clusters contain 200–250 units, not to the subsequent statement about insertions. The paper’s opening section instead calculates enough rDNA for about 130 templates for each ribosomal RNA type per haploid genome; it does not give 200–250 units per cluster. Page numbers are unavailable in our copy. (LLM, conf=0.78)

  Checked by a reasoning model — may be wrong.

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim.

Only a part of rDNA units is transcriptionally active at any time, while the rest, or even the majority of rDNA repeats, are in a repressed state.

“Numerous genetic, microscopic and biochemical studies have demonstrated that only a part of rDNA units is transcriptionally active at any time, while the rest, or even the majority of rDNA repeats, are in a repressed state (6–14).”

• cites [6] Miller, 1969, Visualization of nucleolar genes, Science sourceNot checked — no full text of the source
• cites [7] McKnight, 1976, Ultrastructural patterns of RNA synthesis during early embryogenesis of drosophila melanogaster, Cell sourceNot checked — no full text of the source
• cites [8] Conconi, 1989, Two different chromatin structures coexist in ribosomal RNA genes throughout the cell cycle, Cell sourceNot checked — no full text of the source
• cites [9] Conconi, 1992, Ribosomal gene clusters are uniquely proportioned between open and closed chromatin structures in both tomato leaf cells and exponentially growing suspension cultures, Proc. Natl. Acad. Sci. USA sourceCould not verify

  The indexed copy provides an abstract and references, but no article-body passage addressing how many rDNA units are transcriptionally active or repressed. I searched for the majority of tomato rDNA clusters being in closed chromatin; the matching statement appears only in the abstract, which cannot serve as evidence under this rubric. (LLM, conf=0.72)

  Experimental — accuracy not yet measured
• cites [10] Stancheva, 1997, Chromatin structure and methylation of rat rRNA genes studied by formaldehyde fixation and psoralen cross-linking, Nucleic Acids Res. sourceSupported
• cites [11] Dammann, 1993, Chromatin structures and transcription of rDNA in yeast saccharomyces cerevisiae, Nucleic Acids Res. sourceNot checked — no full text of the source
• cites [12] Parks, 2018, Variant ribosomal RNA alleles are conserved and exhibit tissue-specific expression, Sci. Adv. sourcePartly supported

  The Introduction (page unavailable) describes silencing of distinct rDNA subtypes, and the Results section on expression between organs (page unavailable) notes methylation of nonexpressed rDNA regions. These passages support the existence of active and repressed units, but do not establish that most repeats are repressed or quantify the active fraction at any given time. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [13] Pontvianne, 2013, Subnuclear partitioning of rRNA genes between the nucleolus and nucleoplasm reflects alternative epiallelic states, Genes Dev. sourcePartly supported

  The MET1-dependent CG methylation section reports that approximately half of the rRNA gene pool is silenced in Arabidopsis, supporting the claim that only a portion is active. The paper does not establish that a majority of repeats is repressed; its reported proportion is approximately half. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [14] Rabanal, 2017, Epistatic and allelic interactions control expression of ribosomal RNA gene clusters in arabidopsis thaliana, Genome Biol. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Repressive histone modifications, DNA methylation and nucleosome remodeling are mechanisms responsible for rDNA silencing in yeast and mammalian cells.

“Repressive histone modifications, DNA methylation and nucleosome remodeling have been identified as the mechanisms responsible for rDNA silencing in studies performed mainly in yeast and mammalian cells (see (26–29) for reviews).”

• cites [26] Schofer, 2018, Nucleolus and chromatin, Histochem. Cell Biol. sourceSupported
• cites [27] Hamperl, 2013, Chromatin states at ribosomal DNA loci, Biochim. Biophys. Acta sourceNot checked — no full text of the source
• cites [28] Srivastava, 2016, The epigenetic pathways to ribosomal DNA silencing, Microbiol. Mol. Biol. Rev. sourceSupported
• cites [29] Grummt, 2013, Epigenetic control of RNA polymerase i transcription in mammalian cells, Biochim. Biophys. Acta sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Electron microscopy showed that the low level of R2 transcripts is mainly due to transcriptional repression of the entire rDNA units that contain insertions.

“Electron microscopy using the ‘Miller spreading technique’ (6) showed that the low level of R2 transcripts is mainly due to the transcriptional repression of the entire rDNA units, which contain insertions (51,52).”

• cites [51] Jamrich, 1984, The rare transcripts of interrupted rRNA genes in drosophila melanogaster are processed or degraded during synthesis, EMBO J. sourcePartly supported

  The Results report that nearly all active transcription units in Drosophila bb-8 larvae lack the 5-kb insertion, supporting the inference that most insertion-containing rDNA units are inactive. The paper does not identify the insertion as R2 or establish that repression is the main cause of low R2 transcript levels; it also reports processing or degradation of the rare transcripts produced. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [52] Glatzer, 1979, Lengths of transcribed rDNA repeating units in spermatocytes of drosophila hydei: only genes without an intervening sequence are expressed, Chromosoma sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Within active NORs, some rRNA genes can be silent.

“Moreover, within the active NORs, some rRNA genes can be silent (19,20).”

• cites [19] Kim, 2018, Variation in human chromosome 21 ribosomal RNA genes characterized by TAR cloning and long-read sequencing, Nucleic Acids Res. sourcePartly supported

  The Introduction (page unavailable) says that only 20–50% of rRNA genes are transcriptionally active in most human cells, supporting the point that some can be silent. It does not establish that silent genes occur specifically within active NORs, rather than elsewhere among the NORs. (LLM, conf=0.83)

  Experimental — accuracy not yet measured
• cites [20] Zillner, 2015, Active human nucleolar organizer regions are interspersed with inactive rDNA repeats in normal and tumor cells, Epigenomics sourceNot checked — no full text of the source

May combine two claims.

rRNA genes with insertions produce hundreds or thousands of times fewer transcripts than intact rDNAs, though one work observed only a 10-fold difference.

“In fact, it has been known for several decades that rRNA genes with insertions (hereafter, inserted or interrupted rDNAs) produce hundreds or thousands of times fewer transcripts than intact (uninserted) rDNAs (47–49), though in one work only a 10-fold difference was observed (50).”

• cites [47] Long, 1979, Expression of ribosomal DNA insertions in drosophila melanogaster, Cell sourceNot checked — no full text of the source
• cites [48] Jolly, 1980, Nuclear RNA transcripts from drosophila melanogaster ribosomal RNA genes containing introns, Nucleic Acids Res. sourcePartly supported

  The Results and Discussion report that intron sequences were about 3,000 times less abundant than exon sequences in nuclear RNA from D. melanogaster Kc cells. But the paper says the low abundance could reflect either rare transcription or rapid breakdown; it does not establish that inserted genes produce transcripts 3,000 times less often. The 10-fold observation is attached to a different citation in the citing sentence. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [49] Kidd, 1981, Drosophila melanogaster ribosomal DNA containing type II insertions is variably transcribed in different strains and tissues, J. Mol. Biol. sourceNot checked — no full text of the source
• cites [50] Ye, 2006, Chromatin structure and transcription of the R1- and R2-inserted rRNA genes of drosophila melanogaster, Mol. Cell. Biol. sourceSupported

May combine two claims.

R2 elements do not have their own promoters and therefore can be transcribed only as part of pre-rRNA.

“Along with unusually high specificity of integration, another peculiarity of R2 elements is that they do not have their own promoters and therefore can be transcribed only as a part of pre-rRNA (39–42).”

• cites [39] George, 1999, Conserved features at the 5 end of drosophila R2 retrotransposable elements: implications for transcription and translation, Insect Mol. Biol. sourceNot checked — no full text of the source
• cites [40] Eickbush, 2008, Epigenetic regulation of retrotransposons within the nucleolus of drosophila, Mol. Cell. Biol. sourceNot checked — no full text of the source
• cites [41] Eickbush, 2003, Transcription of endogenous and exogenous R2 elements in the rRNA gene locus of drosophila melanogaster, Mol. Cell. Biol. sourceNot checked — no full text of the source
• cites [42] Eickbush, 2010, R2 retrotransposons encode a self-cleaving ribozyme for processing from an rRNA cotranscript, Mol. Cell. Biol. sourcePartly supported

  The introductory passage says attempts to identify a promoter at the 5′ end of R2 have been unsuccessful and that R2 expression appears to depend on transcription of the rRNA unit. The Discussion describes R2 RNA as processed from an rRNA cotranscript. These passages support the proposed route of transcription, but do not establish that R2 has no promoter or can be transcribed only as part of pre-rRNA. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Some level of R2 expression may be beneficial for the host because the R2 endonuclease can initiate recombination restoring rDNA clusters in cells with decreased rDNA copy number.

“On the other hand, some level of R2 expression may be beneficial for the host, since it has been suggested that the R2 endonuclease can initiate recombination restoring rDNA clusters in cells with decreased rDNA copy number (45,46).”

• cites [45] Hawley, 1989, Recombinational controls of rDNA redundancy in drosophila, Annu. Rev. Genet. sourceNot checked — no full text of the source
• cites [46] Nelson, 2021, The retrotransposon R2 maintains drosophila ribosomal DNA repeats sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Full-length R2 insertions encode a ribozyme that releases the 5′-end of the R2 transcript from the upstream 28S rRNA sequence during the autocatalytic reaction.

“Full-length R2 insertions encode a ribozyme, which releases the 5′-end of the R2 transcript from the upstream 28S rRNA sequence during the autocatalytic reaction (Figure 1A)”

• cites [30] Eickbush, 2015, Integration, regulation, and long-term stability of R2 retrotransposons, Microbiol. Spectr. sourceSupported
• cites [42] Eickbush, 2010, R2 retrotransposons encode a self-cleaving ribozyme for processing from an rRNA cotranscript, Mol. Cell. Biol. sourceSupported

May combine two claims.

Most uninserted rRNA genes are also silenced; psoralen cross-linking revealed that only <10% of the total rDNA units are actively transcribed.

“Furthermore, it was found that most uninserted rRNA genes are also silenced: psoralen cross-linking assay revealed that only <10% of the total rDNA units are actively transcribed (50).”

• cites [50] Ye, 2006, Chromatin structure and transcription of the R1- and R2-inserted rRNA genes of drosophila melanogaster, Mol. Cell. Biol. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Egg knockdown causes about a 40-fold derepression of the telomeric HeT-A element.

“Note that Egg was previously shown to be strongly required for transcriptional silencing of a broad range of TEs in ovaries (84) and its knockdown caused about a 40-fold derepression of telomeric HeT-A element in our analysis (Figure 2A).”

• cites [84] Sienski, 2015, Silencio/CG9754 connects the Piwi-piRNA complex to the cellular heterochromatin machinery, Genes Dev. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

R1 elements occupy from 15% to 67% of rRNA genes, and R2 up to about 30%, depending on D. melanogaster strain.

“R1 elements occupy from 15% to 67% of rRNA genes, and R2 up to ∼ 30% depending on D. melanogaster strain (33).”

• cites [33] Jakubczak, 1992, Turnover of R1 (type I) and R2 (type II) retrotransposable elements in the ribosomal DNA of drosophila melanogaster, Genetics sourcePartly supported

  The “7.5-2.2 kb” section reports R1 insertions in 17–67% of 28S rRNA genes and R2 insertions in 2–28%, varying by strain. That supports the upper bounds and the approximate R2 figure, but not the stated 15% R1 lower bound; the measurements were also limited to X-chromosome rDNA repeats. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Analysis of the proportion and interposition of inserted and uninserted rRNA genes within clusters suggested a transcriptional domains model in which the region lacking R1/R2 insertions is selected to be active while the rest of the locus undergoes heterochromatinization.

“An analysis of the proportion and interposition of inserted and uninserted rRNA genes within clusters suggested a ‘transcriptional domains’ model according to which the region of the rDNA cluster lacking R1/R2 insertions is selected to be transcriptionally active, while the rest of the locus undergoes heterochromatinization (40,65).”

• cites [40] Eickbush, 2008, Epigenetic regulation of retrotransposons within the nucleolus of drosophila, Mol. Cell. Biol. sourceNot checked — no full text of the source
• cites [65] Zhou, 2009, The pattern of R2 retrotransposon activity in natural populations of drosophila simulans reflects the dynamic nature of the rDNA locus, PLos Genet. sourcePartly supported

  Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Maintaining a subset of rRNA genes in an inactive heterochromatic state is thought to be important for ensuring nucleolar structure and preventing recombination between rDNA repeats.

“Second, maintaining a subset of rRNA genes in an inactive heterochromatic state is thought to be important for ensuring nucleolar structure and preventing recombination between rDNA repeats (22–24).”

• cites [22] Peng, 2007, H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability, Nat. Cell Biol. sourceNot checked — no full text of the source
• cites [23] Guetg, 2010, The NoRC complex mediates the heterochromatin formation and stability of silent rRNA genes and centromeric repeats, EMBO J. sourceNot checked — no full text of the source
• cites [24] Kobayashi, 2014, Ribosomal RNA gene repeats, their stability and cellular senescence, Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. sourcePartly supported

  The section “Transcription of rDNA and the role of non-transcribed repeat” (page not provided) says that about half the repeats are inactive and that these units bind condensin and provide space for DNA repair. The copy does not say that keeping those genes in a heterochromatic state ensures nucleolar structure or prevents recombination; it describes condensin's role in repair by homologous recombination. The “Regulation of the number of rDNA repeats” section separately describes Sir2-mediated repression of a promoter that increases recombination. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Run-on analysis indicated that transcription is often terminated within the insertion sequence.

“However, run-on analysis indicated that transcription is often terminated within the insertion sequence (50).”

• cites [50] Ye, 2006, Chromatin structure and transcription of the R1- and R2-inserted rRNA genes of drosophila melanogaster, Mol. Cell. Biol. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

R2 elements are present exclusively at the rDNA locus, whereas R1 insertions have also been found in other regions of the genome.

“R2 elements are of particular interest because they are present exclusively at the rDNA locus, whereas R1 insertions have also been found in other regions of the genome (35–38).”

• cites [35] Kidd, 1980, A DNA segment from d. melanogaster which contains five tandemly repeating units homologous to the major rDNA insertion, Cell sourceNot checked — no full text of the source
• cites [36] Peacock, 1981, Chromosomal distribution of the major insert in drosophila melanogaster 28S rRNA genes, Genet. Res. sourcePartly supported

  The Results and Discussion (page not provided) locate type I insert sequences outside rDNA, including on chromosome 4 and in autosomal heterochromatin, supporting the R1 portion. The Introduction describes type II inserts within 28S rRNA genes but does not establish that they occur exclusively at the rDNA locus, so the R2 portion is not supported. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [37] Browne, 1984, Site specific insertion of a type i rDNA element into a unique sequence in the drosophila melanogaster genome, Nucleic Acids Res. sourcePartly supported

  The Introduction says type II insertions occur in rDNA units, but does not establish that they occur exclusively at the rDNA locus. It describes type I elements outside rDNA, and the Discussion reports a type I insertion in a unique genomic sequence. Thus the R1 portion is supported, but the exclusivity claim for R2 is not established by this copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured
• cites [38] Plata, 2009, Changes in chromatin structure correlate with transcriptional activity of nucleolar rDNA in polytene chromosomes, Chromosoma sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Approximately 20-30% of rDNA repeats in human cell lines are palindromic and noncanonical.

“For instance, approximately 20–30% of rDNA repeats in human cell lines are palindromic and noncanonical (20,25).”

• cites [20] Zillner, 2015, Active human nucleolar organizer regions are interspersed with inactive rDNA repeats in normal and tumor cells, Epigenomics sourceNot checked — no full text of the source
• cites [25] Caburet, 2005, Human ribosomal RNA gene arrays display a broad range of palindromic structures, Genome Res. sourcePartly supported

  The paper identifies palindromic rDNA units as noncanonical, but reports about one-third of units in control human cells, rather than 20–30% (opening summary; Southern blotting analysis, Fig. 3). It reports a higher proportion—about one-half—in Werner syndrome cell lines, so the stated range does not describe all the human cell lines studied. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Differences in chromatin density and histone modifications between inserted and uninserted rDNA units have not been observed in embryonic cells.

“However, differences in chromatin density and histone modifications between inserted and uninserted rDNA units have not been observed in embryonic cells (50).”

• cites [50] Ye, 2006, Chromatin structure and transcription of the R1- and R2-inserted rRNA genes of drosophila melanogaster, Mol. Cell. Biol. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Repression of a fraction of rRNA genes can maintain an optimal level of ribosome production regardless of the rDNA copy number in the genome.

“First, this phenomenon can be useful to maintain an optimal level of ribosome production regardless of the rDNA copy number in the genome (21).”

• cites [21] McStay, 2006, Nucleolar dominance: a model for rRNA gene silencing, Genes Dev. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Drosophila melanogaster lacks UBF homolog, while Tif-IA and the SL1-like complex were described in the fruit fly.

“Drosophila melanogaster lacks UBF homolog, while Tif-IA and the SL1-like complex were described in the fruit fly (68,88).”

• cites [68] Zhang, 2014, Changes in rRNA transcription influence proliferation and cell fate within a stem cell lineage, Science sourcePartly supported

  The passage describing the Udd–TAF1B–TAF1C-like complex (section labeled Abstract; page unavailable) identifies a Drosophila complex analogous to human SL1. The paper also discusses TIF-IA as a Drosophila Pol I regulator. Our copy does not state that Drosophila lacks a UBF homolog, so that part of the claim is unsupported. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [88] Grewal, 2007, Drosophila TIF-IA is required for ribosome synthesis and cell growth and is regulated by the TOR pathway, J. Cell Biol. sourcePartly supported

  The Results and discussion state that D. melanogaster has no obvious UBF homologue, and the Introduction identifies and studies its TIF-IA homologue. Our copy does not describe an SL1-like complex in the fruit fly; the SL1 mention found is in a reference about human RNA polymerase I. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

Repression of inserted rDNA repeats can occur through termination of transcription within the insertion sequence or due to transcriptional silencing of the entire rDNA unit.

“It has been previously shown that the repression of inserted rDNA repeats can occur through termination of transcription within the insertion sequence (50) or due to the transcriptional silencing of the entire rDNA unit (51).”

• cites [50] Ye, 2006, Chromatin structure and transcription of the R1- and R2-inserted rRNA genes of drosophila melanogaster, Mol. Cell. Biol. sourceSupported
• cites [51] Jamrich, 1984, The rare transcripts of interrupted rRNA genes in drosophila melanogaster are processed or degraded during synthesis, EMBO J. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Some rDNA units contain R1/R2 insertions shortened to varying degrees from the 5′-end, formed due to abortive reverse transcription.

“Some rDNA units contain R1/R2 insertions shortened to varying degrees from the 5′-end, formed due to abortive reverse transcription (30,41).”

• cites [30] Eickbush, 2015, Integration, regulation, and long-term stability of R2 retrotransposons, Microbiol. Spectr. sourcePartly supported

  The section “R2 copies are rapidly gained and lost from rDNA loci” describes R2 insertions with 5′ truncations of varying lengths. “Current model of R2 integration” says these arise when reverse transcriptase does not reach the 5′ end of the RNA template. The Introduction places R1 insertions in rDNA, but our copy does not establish the same truncation mechanism for R1. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [41] Eickbush, 2003, Transcription of endogenous and exogenous R2 elements in the rRNA gene locus of drosophila melanogaster, Mol. Cell. Biol. sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

SL1 specifically recognizes and binds rDNA promoter sequences.

“SL1 specifically recognizes and binds rDNA promoter sequences (87).”

• cites [87] Goodfellow, 2013, Basic mechanisms in RNA polymerase i transcription of the ribosomal RNA genes, Subcell. Biochem. sourceSupported

Classified as probably not a substantial claim. May combine two claims.

SL1 complex is known to recognise the rDNA core promoter and enable rRNA transcription start at the 5′ end of the ETS.

“Given that the SL1 complex is known to recognise the rDNA core promoter and enable rRNA transcription start at the 5′ end of the ETS (87), we examined whether rRNA transcription in udd mutants could be aberrant.”

• cites [87] Goodfellow, 2013, Basic mechanisms in RNA polymerase i transcription of the ribosomal RNA genes, Subcell. Biochem. sourcePartly supported

  Section 10.3.1 says SL1 recognizes and binds the rDNA core promoter and is essential for recruiting Pol I to the transcription start site. Section 10.2 also links the core promoter to rRNA transcription initiation. Our copy does not identify that start site as the 5′ end of the ETS, so that location-specific part is not verified. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

Germline knockdown of Su(var)3-9 leads to much more severe oogenesis defects than Egg depletion but also induces a 2-3-fold upregulation of R1 and R2 elements.

“GKD of another HMT, Su(var)3–9 known to be involved in the formation of constitutive heterochromatin and the spreading of the H3K9me3 mark (84–86) led to much more severe defects of oogenesis than Egg depletion but also induced a 2–3-fold upregulation of R1 and R2 elements (Figure 2A).”

• cites [84] Sienski, 2015, Silencio/CG9754 connects the Piwi-piRNA complex to the cellular heterochromatin machinery, Genes Dev. sourceSupported
• cites [85] Brower-Toland, 2009, Multiple SET methyltransferases are required to maintain normal heterochromatin domains in the genome of drosophila melanogaster, Genetics sourceSupported
• cites [86] Lundberg, 2013, HP1a, Su(var)3-9, SETDB1 and POF stimulate or repress gene expression depending on genomic position, gene length and expression pattern in drosophila melanogaster, Nucleic Acids Res. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Severe decline in rRNA synthesis was previously described for the udd1/uddnull mutant.

“Although a severe decline in rRNA synthesis was previously described for the udd1/uddnull mutant (68), our RT-qPCR analysis showed about a 2-fold reduction of the ETS and 18S-ITS1 transcripts in udd1/uddnull ovaries (Figure 4A and Supplementary Figure S7B).”

• cites [68] Zhang, 2014, Changes in rRNA transcription influence proliferation and cell fate within a stem cell lineage, Science sourcePartly supported

  The passage describing Fig. 2E reports little nascent-rRNA incorporation in homozygous uddnull cells, supporting a severe decline in rRNA synthesis in that genotype. It reports impaired ribosome production in udd1/uddnull cells, but does not directly describe a severe decline in rRNA synthesis for that specific genotype. (LLM, conf=0.84)

  Experimental — accuracy not yet measured

May combine two claims.

The histone replacement system abolishes all modifications of H3K9 and mainly manifests itself in derepression of TEs likely due to the loss of H3K9me2/3 marks.

“Although this replacement abolishes all modifications of H3K9, it mainly manifests itself in derepression of TEs likely due to the loss of H3K9me2/3 marks (71).”

• cites [71] Penke, 2016, Direct interrogation of the role of H3K9 in metazoan heterochromatin function, Genes Dev. sourcePartly supported

  The Abstract and Results report that H3K9R replacement leaves most protein-coding expression relatively normal while derepressing most transposon families; the Results link transposon activation to loss of HP1a and a repressive chromatin environment. The claim that it abolishes all H3K9 modifications is too broad: the Results report unchanged H3K9me3 signal on chromosome arms, potentially from replication-independent H3.3. Our copy provides no page numbers for these passages. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Disruption of SL1-like compromised rRNA synthesis and development of ovarian germ cells.

“It has been shown that the disruption of SL1-like compromised rRNA synthesis and development of ovarian germ cells (68).”

• cites [68] Zhang, 2014, Changes in rRNA transcription influence proliferation and cell fate within a stem cell lineage, Science sourceSupported

Classified as probably not a substantial claim. May combine two claims.

Drosophila SL1-like was described as an analog of the mammalian SL1 complex, which promotes rDNA transcription by recognizing and binding the rDNA core promoter sequence and recruiting Pol I.

“Drosophila SL1-like was described as an analog of the mammalian SL1 complex (68), which promotes rDNA transcription by recognizing and binding the rDNA core promoter sequence and recruiting Pol I (87).”

• cites [68] Zhang, 2014, Changes in rRNA transcription influence proliferation and cell fate within a stem cell lineage, Science sourceSupported
• cites [87] Goodfellow, 2013, Basic mechanisms in RNA polymerase i transcription of the ribosomal RNA genes, Subcell. Biochem. sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Studies on mammalian cells show that rDNA repeats in the nucleolus form loops connecting promoter and termination regions or more complex structures, in which the transcribed rDNA region is wrapped around the core formed by SL1.

“Studies on mammalian cells show that rDNA repeats in the nucleolus form loops connecting promoter and termination regions (91,92) or more complex structures, in which the transcribed rDNA region is wrapped around the core formed by SL1 (93).”

• cites [91] Nemeth, 2008, Epigenetic regulation of TTF-I-mediated promoter-terminator interactions of rRNA genes, EMBO J. sourceSupported
• cites [92] Shiue, 2009, c-Myc induces changes in higher order rDNA structure on stimulation of quiescent cells, Oncogene sourceNot checked — no full text of the source
• cites [93] Denissov, 2011, A model for the topology of active ribosomal RNA genes, EMBO Rep. sourcePartly supported

  The ‘core-helix model’ section proposes that the transcribed rDNA region wraps around an SL1-based anchoring core. The citing sentence presents that proposed structure as something studies have shown, whereas this paper describes the wrapping as a model, not a demonstrated arrangement. Citation 93 attaches to this core-helix clause, rather than the preceding loop claim. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

A substantial number of rDNA units in the D. melanogaster genome contain insertions of R1 and R2 retrotransposons usually causing these units to be expressed at very low levels.

“It has been known for several decades that a substantial number of rDNA units in the D. melanogaster genome contain insertions of R1 and R2 retrotransposons usually causing these units to be expressed at very low levels (47,48,51).”

• cites [47] Long, 1979, Expression of ribosomal DNA insertions in drosophila melanogaster, Cell sourceNot checked — no full text of the source
• cites [48] Jolly, 1980, Nuclear RNA transcripts from drosophila melanogaster ribosomal RNA genes containing introns, Nucleic Acids Res. sourcePartly supported

  The Introduction (page unavailable) says that about 49% of rDNA repeats in the female-derived cells studied have type I introns. The Abstract (page unavailable) reports very low abundance of long intron-containing transcripts, but does not establish that the insertions cause low expression; it also raises rapid processing as a possibility. Our copy does not identify the introns as R1 and R2 retrotransposons. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [51] Jamrich, 1984, The rare transcripts of interrupted rRNA genes in drosophila melanogaster are processed or degraded during synthesis, EMBO J. sourcePartly supported

  The Results report that about half the rRNA genes in the D. melanogaster bb-8 mutant contain a 5-kb intervening sequence and that most such genes appear transcriptionally inactive. The paper does not identify those sequences as R1 and R2 retrotransposons; it says their nature cannot be unequivocally identified. Its finding in bb-8 larvae also does not establish the claimed prevalence across the D. melanogaster genome generally. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

The R2 element integrates exclusively into the rDNA locus and is capable to be transcribed only as part of pre-rRNA.

“The R2 element integrates exclusively into the rDNA locus and is capable to be transcribed only as part of pre-rRNA (39–42).”

• cites [39] George, 1999, Conserved features at the 5 end of drosophila R2 retrotransposable elements: implications for transcription and translation, Insect Mol. Biol. sourceNot checked — no full text of the source
• cites [40] Eickbush, 2008, Epigenetic regulation of retrotransposons within the nucleolus of drosophila, Mol. Cell. Biol. sourceNot checked — no full text of the source
• cites [41] Eickbush, 2003, Transcription of endogenous and exogenous R2 elements in the rRNA gene locus of drosophila melanogaster, Mol. Cell. Biol. sourceNot checked — no full text of the source
• cites [42] Eickbush, 2010, R2 retrotransposons encode a self-cleaving ribozyme for processing from an rRNA cotranscript, Mol. Cell. Biol. sourcePartly supported

  The introduction (page unavailable) says that all active R2 copies reported to date occupy a conserved site in the 28S rRNA gene, and that R2 expression appears to depend on transcription of the rDNA unit containing it. The Results detect 28S–R2 cotranscripts, but the paper does not establish that every R2 insertion is in rDNA or that R2 can be transcribed only as part of pre-rRNA. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Egg is involved in de novo H3K9me3 establishment, while Su(var)3-9 is required for H3K9me3 spreading.

“which may be due to the fact that Egg is involved in de novo H3K9me3 establishment, while Su(var)3–9 is required for H3K9me3 spreading (84)”

• cites [84] Sienski, 2015, Silencio/CG9754 connects the Piwi-piRNA complex to the cellular heterochromatin machinery, Genes Dev. sourcePartly supported

  The Results section, “Distinct roles of SetDB1 and Su(var)3-9 at piRNA targeted TE insertions,” proposes that Egg/SetDB1 initiates heterochromatin formation and Su(var)3-9 assists H3K9me3 spreading at piRNA-targeted TE insertions. It reports reduced, not abolished, spreading after Su(var)3-9 depletion. The claim’s unqualified statement that Su(var)3-9 is required for spreading is stronger and broader than the paper’s finding. (LLM, conf=0.85)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Three components of SL1-like have been identified: TAF1b and TAF1c-like subunits, which have orthologs in mammals, and a Drosophila-specific small subunit Udd.

“Three components of SL1-like have been identified: TAF1b and TAF1c-like subunits, which have orthologs in mammals, and a Drosophila-specific small subunit Udd (68).”

• cites [68] Zhang, 2014, Changes in rRNA transcription influence proliferation and cell fate within a stem cell lineage, Science sourcePartly supported

  The passage describing the protein-identification experiments (section labeled Abstract; page unavailable) identifies Udd, TAF1B, and a TAF1C-like protein as the three components and describes the latter two as homologous or similar to human proteins. The copy does not establish that Udd is small or specific to Drosophila. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

This may also be due to the Pol I pausing signal, the presence of which at the R1 5′-end was recently proposed.

“This may also be due to the Pol I pausing signal, the presence of which at the R1 5′-end was recently proposed (54).”

• cites [54] Raje, 2018, R1 retrotransposons in the nucleolar organizers of drosophila melanogaster are transcribed by RNA polymerase i upon heat shock, Transcription sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

H3K27me3 and H4K20me3 marks have been shown to be involved in rDNA silencing in mammalian cells.

“However, the levels of H3K27me3 and H4K20me3 marks, which have also been shown to be involved in rDNA silencing in mammalian cells (94,95) did not change significantly for any of the analyzed sequences (Supplementary Figure S10B and C).”

• cites [94] Bierhoff, 2014, Quiescence-induced lncrnas trigger H4K20 trimethylation and transcriptional silencing, Mol. Cell sourceNot checked — no full text of the source
• cites [95] Xie, 2012, The chromatin remodeling complex NuRD establishes the poised state of rRNA genes characterized by bivalent histone modifications and altered nucleosome positions, Proc. Natl. Acad. Sci. USA sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Knockdowns of SUMO and SUMO ligase Ubc9 induce dramatic derepression of inserted rDNA units and, probably, activation of silent rDNA repeats lacking R1/R2 insertions.

“In parallel with our investigations, it was shown that knockdowns of SUMO (Small Ubiquitin-like Modifier) and SUMO ligase Ubc9 induce dramatic derepression of inserted rDNA units and, probably, activation of silent rDNA repeats lacking R1/R2 insertions (108).”

• cites [108] Luo, 2020, Repression of interrupted and intact rDNA by the SUMO pathway in drosophila melanogaster, Elife sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

Silent rRNA genes in mammalian cells have hypermethylated DNA and regular nucleosomes carrying repressive histone modifications, such as H3K9me3, H4K20me3 and H3K27me3.

“Silent rRNA genes in mammalian cells are known to have hypermethylated DNA and regular nucleosomes carrying repressive histone modifications, such as H3K9me3, H4K20me3 and H3K27me3 (94,95,102,103)”

• cites [94] Bierhoff, 2014, Quiescence-induced lncrnas trigger H4K20 trimethylation and transcriptional silencing, Mol. Cell sourceNot checked — no full text of the source
• cites [95] Xie, 2012, The chromatin remodeling complex NuRD establishes the poised state of rRNA genes characterized by bivalent histone modifications and altered nucleosome positions, Proc. Natl. Acad. Sci. USA sourcePartly supported

  The Discussion describes silent rRNA genes as methylated and heterochromatic. In “CHD4/NuRD and CSB Cooperate in Pol I Transcription Activation,” H4K20me3 and H3K27me3 are associated with the silent-gene nucleosome position, but H3K9me3 occurs at both active and silent positions. The cited passages do not establish that silent genes have regular nucleosomes. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [102] Zhou, 2002, The chromatin remodeling complex NoRC targets HDAC1 to the ribosomal gene promoter and represses RNA polymerase i transcription, EMBO J. sourcePartly supported

  The Introduction says inactive rDNA copies contain nucleosomes, and the Discussion reports CpG methylation and H3 lysine-9 methylation at silent ribosomal genes. The Discussion does not specify H3K9 trimethylation or report H4K20me3 or H3K27me3; it also says the DNA is methylated without characterizing it as hypermethylated. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [103] Santoro, 2002, The nucleolar remodeling complex NoRC mediates heterochromatin formation and silencing of ribosomal gene transcription, Nat. Genet. sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The loss of SUMO leads to a stronger derepression of R1 compared to R2 elements and increases the total pre-rRNA production.

“In particular, the loss of SUMO leads to a stronger derepression of R1 compared to R2 elements and increases the total pre-rRNA production (108).”

• cites [108] Luo, 2020, Repression of interrupted and intact rDNA by the SUMO pathway in drosophila melanogaster, Elife sourceSupported

May combine two claims.

The effects upon SUMO knockdown cannot be explained solely through Udd or SL1-like functions, but apparently represent a more extensive release of rDNA transcription.

“Although Udd was found among nucleolar targets of SUMOylation (108), the effects upon SUMO knockdown cannot be explained solely through Udd or SL1-like functions, but apparently represent a more extensive release of rDNA transcription.”

• cites [108] Luo, 2020, Repression of interrupted and intact rDNA by the SUMO pathway in drosophila melanogaster, Elife sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

In D. melanogaster, heterochromatin components are implicated in maintaining the nucleolar structure and preventing recombination between rDNA repeats, as well as in nucleolar dominance.

“In D. melanogaster, heterochromatin components have been shown to be implicated in maintaining the nucleolar structure and preventing recombination between rDNA repeats (22), as well as in nucleolar dominance, a phenomenon whereby an entire rDNA cluster is silenced (104).”

• cites [22] Peng, 2007, H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability, Nat. Cell Biol. sourceNot checked — no full text of the source
• cites [104] Warsinger-Pepe, 2020, Regulation of nucleolar dominance in drosophila melanogaster, Genetics sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Loss of a ribosome assembly factor Nopp140 leads to both reduced rRNA synthesis and R2 activation.

“Our assumptions are consistent with previous finding that loss of a ribosome assembly factor Nopp140 leads to both reduced rRNA synthesis (101) and R2 activation (64).”

• cites [101] Neumüller, 2013, Conserved regulators of nucleolar size revealed by global phenotypic analyses, Sci. Signal sourceSupported
• cites [64] He, 2015, Deletion of drosophila nopp140 induces subcellular ribosomopathies, Chromosoma sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

Binding of Su(var)3-9 with chromatin partially depends on Egg in ovarian germ cells.

“and its binding with the chromatin partially depends on Egg in ovarian germ cells (106).”

• cites [106] Maksimov, 2019, Binding of SU(VAR)3-9 partially depends on SETDB1 in the chromosomes of drosophila melanogaster, Cells sourceSupported

May combine two claims.

The w1118 female genome contains a total of 206 rDNA genes, of which 20 (about 10%) have R2 insertions.

“We calculated that the w1118 female genome contains a total of 206 rDNA genes, of which 20 (∼10%) have R2 insertions.”

May combine two claims.

The average pre-rRNA transcription level per rDNA repeat is higher than transcription of any protein-coding gene and approximately 100 and 30 times higher than that of R1 or R2 elements, respectively.

“Our analysis showed that the average pre-rRNA transcription level per rDNA repeat is higher than transcription of any protein-coding gene, and approximately 100 and 30 times higher than that of R1 or R2 elements, respectively (Figure 1B).”

May combine two claims.

In both udd0683-G4/uddnull and udd1/uddnull ovaries, FISH signals were faint in germ cells of germarium and early egg chamber stages, then gradually increased as oogenesis progressed and reached a maximum in nurse cells at the latest stages present in mutants.

“Remarkably, in both udd0683-G4/uddnull and udd1/uddnull ovaries FISH signals were faint in germ cells of germarium and early egg chamber stages, then gradually increased as oogenesis progressed and reached a maximum in nurse cells at the latest stages present in mutants (Figure 3С and Supplementary Figure S6A).”

May combine two claims.

Nucleoli in nurse cells of udd mutants were less branched and appeared as large intranuclear bodies.

“However, the nucleoli in nurse cells of udd mutants were less branched and appeared as large intranuclear bodies (Figure 3D–F).”

May combine two claims.

All other tested udd mutant flies (udd1/udd1, udd1/uddnull, udd0683-G4/uddnull and udd0683-G4/udd0683-G4) were sterile and had reduced ovaries and testes.

“All other tested udd mutant flies (udd1/udd1, udd1/uddnull, udd0683-G4/uddnull and udd0683-G4/ udd0683-G4) were sterile and had reduced ovaries and testes (in accordance with (68)) but did not display any morphological body defects known to be associated with significantly decreased ribosome production (minute-like phenotypes (89)).”

May combine two claims.

R2 transcripts tended to accumulate in the central part of the nucleolus, while pre-rRNA was localized mainly at its periphery.

“Interestingly, R2 transcripts tended to accumulate in the central part of the nucleolus, while pre-rRNA was localized mainly at its periphery (Figure 3F).”

May combine two claims.

The amount of IGS-ETS RNA was still about 25-fold lower than the amount of transcripts corresponding to the beginning of the ETS in the udd0683-G4/uddnull ovaries, whereas for the udd1/uddnull mutant this difference was greater.

“Nevertheless, the amount of IGS-ETS RNA was still about 25-fold lower than the amount of transcripts corresponding to the beginning of the ETS in the udd0683-G4/uddnull ovaries, whereas for the udd1/uddnull mutant this difference was greater (Figure 4A).”

May combine two claims.

udd0683-G4/uddnull ovaries had a more normal phenotype and retained oogenesis up to stage 11.

“udd0683-G4/uddnull ovaries had a more normal phenotype and retained oogenesis up to stage 11 (Figure 3С and Supplementary Figure S5).”

May combine two claims.

Udd was largely released from the nucleolus and found in the cytoplasm of ActD-treated cells.

“Interestingly, Udd was largely released from the nucleolus and found in the cytoplasm of ActD-treated cells (Supplementary Figure S11A).”

May combine two claims.

We also found Udd enrichment in the region of rDNA transcription termination on the border of 28S and IGS sequences, as well as in the IGS.

“We also found Udd enrichment in the region of rDNA transcription termination on the border of 28S and IGS sequences, as well as in the IGS (Figure 6A and Supplementary Figure S9).”

May combine two claims.

ChIP-qPCR and ChIP-seq showed high Udd enrichment at the rDNA promoter region (ETS) with the most pronounced peak at the border of IGS and ETS.

“Consistent with the previous report (68), ChIP-qPCR and ChIP-seq showed high Udd enrichment at the rDNA promoter region (ETS on Figure 6A) with the most pronounced peak at the border of IGS and ETS (dotted line in Figure 6B).”

May combine two claims.

We detected rDNA units containing the 3′-UTR of the R1 element by PCR of udd0683-G4/uddnull genomic DNA and demonstrated by RT-qPCR that these units were 200-fold upregulated in udd0683-G4/uddnull ovaries.

“Indeed, we detected rDNA units containing the 3′-UTR of the R1 element by PCR of udd0683-G4/uddnull genomic DNA and demonstrated by RT-qPCR that these units were 200-fold upregulated in udd0683-G4/uddnull ovaries (Supplementary Figure S8).”

May combine two claims.

RNA-seq showed that a short region corresponding to the 3′-UTR of the R1 element was 200-300-fold derepressed in udd0683-G4/uddnull ovaries, whereas the level of transcripts derived from the most of this element increased much less drastically.

“However, RNA-seq showed that a short region corresponding to the 3′-UTR of the R1 element was 200–300-fold derepressed in udd0683-G4/uddnull ovaries, whereas the level of transcripts derived from the most of this element increased much less drastically (Figure 5C, bottom panel).”

May combine two claims.

ActD treatment did not lead to cell death and block of DNA replication.

“We detected cessation of rRNA synthesis in ActD-treated cells, which was accompanied by a reduction of a nucleolar area occupied by fibrillarin (Supplementary Figure S11A and B) but did not lead to cell death and block of DNA replication (Supplementary Figure S11C and D).”

May combine two claims.

A similar pattern of Udd distribution along rDNA units was observed in both ovaries and carcasses, whereas enrichment levels were lower in carcasses.

“A similar pattern of Udd distribution along rDNA units was observed in both ovaries and carcasses, whereas enrichment levels were lower in carcasses (Supplementary Figure S9), which may reflect a higher rate of rRNA synthesis in ovarian cells.”

May combine two claims.

We detected cessation of rRNA synthesis in ActD-treated cells, which was accompanied by a reduction of a nucleolar area occupied by fibrillarin.

“We detected cessation of rRNA synthesis in ActD-treated cells, which was accompanied by a reduction of a nucleolar area occupied by fibrillarin (Supplementary Figure S11A and B) but did not lead to cell death and block of DNA replication (Supplementary Figure S11C and D).”

May combine two claims.

In contrast to the previous report, we did not find a dramatic decrease in the total pre-rRNA derived from both interrupted and intact rDNA units in the ovaries lacking SL1-like components, except for a 2-fold reduction in udd1/uddnull mutants.

“In contrast to the previous report (68), we did not find a dramatic decrease in the total pre-rRNA derived from both interrupted and intact rDNA units in the ovaries lacking SL1-like components, except for a 2-fold reduction in udd1/uddnull mutants (Figure 4, Supplementary Figure S7).”

May combine two claims.

Intact rRNA genes can be selected based on a transcriptional feedback loop, whereas less effectively transcribed rDNA copies undergo heterochromatinization.

“Overall, we propose that intact rRNA genes can be selected based on a transcriptional feedback loop, whereas less effectively transcribed rDNA copies undergo heterochromatinization.”

• AssumptionRests on an assumption: It remains generally unknown how cellular machineries select particular rRNA genes to establish their activation or repression.

Classified as probably not a substantial claim.

R2 derepression may serve as an adaptive response to compromised rRNA synthesis.

“We also propose that R2 derepression may serve as an adaptive response to compromised rRNA synthesis.”

• Also stated in: § INTRODUCTION · hedged

  “We also hypothesize that enhanced R2 expression due to the defects of SL1-like can be a part of an adaptive response to reduced rRNA production.”

May combine two claims.

Chromatin marks are non-uniformly distributed along a single rDNA repeat, and promoter regions of rDNA genes are depleted in heterochromatin marks.

“This result indicates that chromatin marks are non-uniformly distributed along a single rDNA repeat and promoter regions of rDNA genes are depleted in heterochromatin marks.”

Probably not a substantial claim: may not pass S3 (load-bearing).

R1 and R2 elements colocalize with condensed chromatin in the nucleolus of salivary gland cells.

“Nevertheless, another report found the colocalization of R1 and R2 elements with condensed chromatin in the nucleolus of salivary gland cells (38).”

• cites [38] Plata, 2009, Changes in chromatin structure correlate with transcriptional activity of nucleolar rDNA in polytene chromosomes, Chromosoma sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

For each ChIP-qPCR value, 3 to 7 biological replicates were analyzed.

“For each value, 3 to 7 biological replicates were analyzed.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Nucleoli in nurse cells develop into branched structures that are different from the ordinary spherical nucleoli of diploid cells.

“It is noteworthy that nucleoli in nurse cells develop into branched structures that are different from the ordinary spherical nucleoli of diploid cells (67).”

• cites [67] Dapples, 1970, The development of the nucleolus of the ovarian nurse cell of drosophila melanogaster, Z. Zellforsch. Mikrosk. Anat. sourceNot checked — no full text of the source

Classified as probably not a substantial claim.

Given the increased contribution of inserted rDNA unit transcription to the total pre-rRNA pool, intact rRNA genes may be downregulated upon knockdowns.

“Thus, given the increased contribution of inserted rDNA unit transcription to the total pre-rRNA pool, intact rRNA genes may be downregulated upon knockdowns.”

• AssumptionThe authors state this as an assumption: Transcription of intact rRNA genes may decrease to some extent upon all examined mutations and knockdowns of SL1-like subunits.

May combine two claims.

Analyzed udd mutants mostly retain normal rRNA transcription start site, while display elevated IGS RNA synthesis and strong upregulation of R1 and notably R2 elements.

“Thus, analyzed udd mutants mostly retain normal rRNA transcription start site, while display elevated IGS RNA synthesis and strong upregulation of R1 and notably R2 elements.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The rp49 mRNA level was not significantly changed in RNA-seq datasets including those for HP1a GKD and udd mutant ovaries.

“RT-qPCR values were normalized to the rp49 mRNA level, which according to our observations was not significantly changed in a number of RNA-seq data sets, including those for HP1a GKD and udd mutant ovaries.”

Probably not a substantial claim: may not pass S3 (load-bearing).

NGS libraries were prepared in duplicates.

“NGS libraries were prepared in duplicates using MGIEasy RNA Library Prep Set V3.1 (MGI Tech Co. Ltd.) according to the manufacturer’s recommendations.”

Classified as probably not a substantial claim.

Transcription of rRNA genes in mammals requires upstream binding factor (UBF), the RRN3 (Tif-IA) protein and the core pre-initiation complex SL1, consisting of TBP and TAFs.

“Transcription of rRNA genes in mammals requires several specific basal factors: upstream binding factor (UBF), the RRN3 (Tif-IA) protein and the core pre-initiation complex SL1, consisting of the TATA-box binding factor (TBP) and TBP-associated factors (TAFs).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Actinomycin D at 1 μg/ml did not lead to complete block of nascent transcription in the nucleolus.

“ActD at lower concentration (1 μg/ml) did not lead to complete block of nascent transcription in the nucleolus.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Formaldehyde cross-linking immediately after manual ovary dissection without freezing is important to obtain high enrichments in ChIP.

“Formaldehyde cross-linking was performed immediately after manual ovary dissection without freezing, that was found to be important to obtain high enrichments.”

May combine two claims.

Normally repression of R2-inserted rRNA genes occurs both by silencing of the entire rDNA unit and by interruption of transcription in the R2 body, while udd mutations release the repression only on the level of the entire rDNA repeat possibly affecting the transcription initiation step.

“Altogether, these results suggest that normally repression of R2-inserted rRNA genes occurs both by silencing of the entire rDNA unit and by interruption of transcription in the R2 body, while udd mutations release the repression only on the level of the entire rDNA repeat possibly affecting the transcription initiation step.”

May combine two claims.

Lack of Udd strongly enhances transcription across R2-inserted rDNA unit but does not prevent transcriptional decline within the R2 body.

“Thus, the lack of Udd strongly enhances transcription across R2-inserted rDNA unit but does not prevent transcriptional decline within the R2 body.”

Probably not a substantial claim: may not pass S3 (load-bearing).

At least three biological replicates of RNA and genomic DNA were used.

“At least three biological replicates of RNA and genomic DNA were used.”

May combine two claims.

Effect of Udd on R2-inserted rDNA units is independent of the insertion length and therefore is unlikely to be determined by any specific nucleotide motifs within the R2 sequence, or at least within its most part.

“Thus, effect of Udd on R2-inserted rDNA units is independent of the insertion length and therefore is unlikely to be determined by any specific nucleotide motifs within the R2 sequence, or at least within its most part.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

PCR products were verified by gel electrophoresis and in some cases by Sanger sequencing.

“PCR-products were verified by gel electrophoresis and in some cases by Sanger sequencing.”

May combine two claims.

The absolute level of 28S-5′R2 cotranscripts is lower than that of 5′ R2 body transcripts because R2 RNA can self-cleave from the 28S rRNA sequence.

“Note that the absolute level of 28S-5′R2 cotranscripts is lower than that of 5′ R2 body transcripts (Figure 5A) because R2 RNA can self-cleave from the 28S rRNA sequence.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

A stage 14 oocyte contains approximately 2 × 10^10 ribosomes, the vast majority of which are produced in the nurse cells during stages 7 through 10.

“It has been calculated that a stage 14 oocyte contains approximately 2 × 1010 ribosomes (66), the vast majority of which are produced in the nurse cells during stages 7 through 10 (67).”

• cites [66] Klug, 1970, Oogenesis in the suppressor of hairy-wing mutant of drosophila melanogaster. II. Nucleolar morphology and in vitro studies of RNA protein synthesis, J. Exp. Zool. sourceNot checked — no full text of the source
• cites [67] Dapples, 1970, The development of the nucleolus of the ovarian nurse cell of drosophila melanogaster, Z. Zellforsch. Mikrosk. Anat. sourceNot checked — no full text of the source

May combine two claims.

Udd and other subunits of SL1-like can serve both as transcription initiation factors and components of a hypothetical repressor complex interacted with silent rDNA units.

“Based on our results, it is possible that Udd and other subunits of SL1-like can serve both as transcription initiation factors and components of a hypothetical repressor complex interacted with silent rDNA units.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Lower enrichment in carcasses may reflect a higher rate of rRNA synthesis in ovarian cells.

“A similar pattern of Udd distribution along rDNA units was observed in both ovaries and carcasses, whereas enrichment levels were lower in carcasses (Supplementary Figure S9), which may reflect a higher rate of rRNA synthesis in ovarian cells.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

This can be interpreted as the formation of a spatial loop or as a binding of the SL1-like to putative IGS promoters.

“This can be interpreted as the formation of a spatial loop or as a binding of the SL1-like to putative IGS promoters.”

May combine two claims.

Complete loss of these proteins is lethal, and the analyzed effects are caused by a partial impairment of the SL1-like complex.

“Apparently, the complete loss of these proteins is lethal, and the analyzed effects are caused by a partial impairment of the SL1-like complex.”

May combine two claims.

Reduction of the H3K9me3 level in rRNA genes in the udd mutant ovaries can be caused by their transcriptional activation, and vice versa repressive chromatin marks can normally be recruited to rDNA units as a consequence of their transcriptional silencing.

“Our observations suggest that the reduction of the H3K9me3 level in rRNA genes in the udd mutant ovaries can be caused by their transcriptional activation, and vice versa repressive chromatin marks can normally be recruited to rDNA units as a consequence of their transcriptional silencing.”

Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based). May combine two claims.

Other factors such as deposition of active chromatin marks, co-transcriptional rRNA processing, and phase separation could also be involved in the positive feedback loop, promoting the accumulation of components of the Pol I transcription apparatus on intact rDNA units.

“Other factors such as deposition of active chromatin marks, co-transcriptional rRNA processing, and phase separation could also be involved in the positive feedback loop, promoting the accumulation of components of the Pol I transcription apparatus on intact rDNA units.”

May combine two claims.

Some rRNA genes lacking R1/R2 insertions are also normally silenced and heterochromatinized, whereas in udd mutants they may be transcriptionally activated and lose heterochromatin marks in a similar way to R2-inserted rDNA units.

“This result suggests that some rRNA genes lacking R1/R2 insertions are also normally silenced and heterochromatinized, whereas in udd mutants they may be transcriptionally activated and lose heterochromatin marks in a similar way to R2-inserted rDNA units.”

May combine two claims.

Truncated R1 insertions are much more strongly derepressed in udd mutants when compared to full-length R1 insertions suggesting that the R1 sequence upstream of the 3′-UTR may contain a putative silencer element, which prevents the complete activation of full-length R1 elements in udd mutants.

“Thus, the truncated R1 insertions are much more strongly derepressed in udd mutants when compared to full-length R1 insertions suggesting that the R1 sequence upstream of the 3′-UTR may contain a putative silencer element, which prevents the complete activation of full-length R1 elements in udd mutants.”

• AssumptionThe authors state this as an assumption: The effect of higher 3′-UTR expression can be attributed to short insertions of the R1 solo 3′-UTR lacking the R1 body.

Classified as probably not a substantial claim.

ActD nonspecifically blocks all types of RNA polymerase.

“ActD nonspecifically blocks all types of RNA polymerase (96).”

• cites [96] Bensaude, 2011, Inhibiting eukaryotic transcription: which compound to choose? How to evaluate its activity, Transcription sourceNot checked — no full text of the source

May combine two claims.

Repressive chromatin can be rapidly established on transcriptionally inactive rDNA units that supports the notion that the difference in H3K9me3 levels between inserted and intact rRNA genes can be a consequence of their distinct transcriptional states.

“Thus, repressive chromatin can be rapidly established on transcriptionally inactive rDNA units that supports the notion that the difference in H3K9me3 levels between inserted and intact rRNA genes can be a consequence of their distinct transcriptional states.”

Probably not a substantial claim: may not pass S3 (load-bearing).

It is conceivable that in ChIP experiments some cross-linking can occur between proteins associated with the promoter and pre-rRNA-coding part of the same rDNA unit.

“Hence, it is conceivable that in ChIP experiments some cross-linking can occur between proteins associated with the promoter and pre-rRNA-coding part of the same rDNA unit.”

May combine two claims.

Transcription of inserted rDNA units normally can be less efficiently initiated than that of intact ones and this repression is released in udd mutants.

“This result suggests that transcription of inserted rDNA units normally can be less efficiently initiated than that of intact ones and this repression is released in udd mutants.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Loss of piRNA-binding protein Piwi on a specific genetic background may cause general transcriptional repression of the rDNA cluster, which leads to partial removal of Udd from the nucleoli of nurse cells.

“At the same time, the loss of piRNA-binding protein Piwi on a specific genetic background may cause general transcriptional repression of the rDNA cluster, which leads to partial removal of Udd from the nucleoli of nurse cells (107).”

• cites [107] Fefelova, 2017, Participation of the piRNA pathway in recruiting a component of RNA polymerase i transcription initiation complex to germline cell nucleoli, Mol. Biol. (Mosk) sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

The observed decrease of H3K9me3 mark at uninserted 28S region in udd mutants can be owing to the derepression of these units.

“The observed decrease of H3K9me3 mark at uninserted 28S region in udd mutants (Figure 6C) can be owing to the derepression of these units.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Our findings parallel with the results revealing the coexistence of methylated and unmethylated rDNA units within the same cluster in human cells.

“Our findings parallel with the results revealing the coexistence of methylated and unmethylated rDNA units within the same cluster using FISH and immunostaining of single DNA fibers in human cells (20).”

• cites [20] Zillner, 2015, Active human nucleolar organizer regions are interspersed with inactive rDNA repeats in normal and tumor cells, Epigenomics sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

The second model better interprets the extremely high upregulation of the R2 element observed in udd0683-G4/uddnull ovaries.

“This model better interprets an extremely high upregulation of the R2 element observed in udd0683-G4/uddnull ovaries.”

May combine two claims.

Heterochromatin marks are deposited on less effectively transcribed rDNA units, strengthening their repression, whereas transcriptional activation can remove the repressive chromatin modifications.

“Altogether, we propose that heterochromatin marks are deposited on less effectively transcribed rDNA units, strengthening their repression, whereas transcriptional activation can remove the repressive chromatin modifications (see model in Supplementary Figure S12).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The transcriptionally inert rDNA genes associated with HP1 have been demonstrated inside active human NORs by super-resolution microscopy.

“Moreover, the transcriptionally inert rDNA genes associated with HP1 have been recently demonstrated inside active human NORs by super-resolution microscopy (105).”

• cites [105] Yao, 2019, Nascent Pre-rRNA sorting via phase separation drives the assembly of dense fibrillar components in the human nucleolus, Mol. Cell sourceNot checked — no full text of the source