Chromatin Structure and Transcription of the R1- and R2-Inserted rRNA Genes of Drosophila melanogaster

“Previous reports have suggested that the rDNA locus can serve as an ideal niche for the long-term survival of TEs [43], as seen in several organisms [44-46].”

“Finally, various assays of transcriptionally active chromatin structure suggested that <10% of the rDNA units were being transcribed (Ye and Eickbush 2006).”

“This is an important point because only a limited number of the rDNA units within the locus, possibly only 30 to 40 units, are transcriptionally active (8, 38, 40) and thus only a small percentage of the R2 copies are transcribed.”

“Diminished capacity for transcribing to the end of the gene may also explain the reduced rRNA synthesis of Drosophila rDNA inserted into retrotransposable elements (Ye and Eickbush 2006).”

“The chromatin associated with R2 insertions contained the typical epigenetic marks linked to heterochromatin (106).”

“R1 and R2 retrotransposable elements selectively insert into the 28S rRNA gene, and inserted rDNA units are transcriptionally repressed (Ye and Eickbush, 2006).”

“dH1 depletion causesDNAdamage and genomic instability that, at least in part, is the consequence of deregulated expression of the rDNA locus due to activation of R1/R2 retrotransposons that are inserted in around 60% of rDNA units (30,48).”

“On: Tue, 25 Apr 2017 14:08:23 suggested that some rDNA-specific non-LTR elements are transcribed by RNA polymerase I, not by RNA polymerase II (96).”

“The presence of hybridizing RNA species longer than 3,600 nt, most readily seen in line 89, is consistent with suggestions that the R2 transcripts were processed from cotranscripts with the 28S gene (9, 13, 18, 19, 25, 43).”

“The R1 and R2 elements insert within the transcribed portion of the rDNA repeat unit and strongly reduce transcription of the unit by stimulating early tran- scriptional termination (Ye and Eickbush 2006).”

“The second predicts that non-LTR retroposons are expressed via co-transcription with an interrupted gene, though to the best of our knowledge, this has yet to be proven experimentally (Eickbush et al., 2000; Malik and Eickbush, 2000; Ye and Eickbush, 2006).”

“Second, the size of the rDNA locus is sufficiently large that only a fraction of the rDNA units encoded by an organism are transcribed at any one time [23,36–39].”

“Generally, rDNA genes with R1 and R2 insertions exhibit very low levels of transcription compared to normal uninserted rDNA genes (Jamrich andMiller 1984; Ye and Eickbush 2006; Eickbush and Eickbush 2015).”

“The number of rDNA units required to supply rRNA in Drosophila has been estimated to be 35–50 units [4,25,26].”

“Possible functions of these sequences include transcription enhancers, transcription terminators to prevent the formation of aberrant rRNA read-through transcripts, or binding sequences to recruit epigenetic modifiers [34-36].”

“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).”

“…site in the gene encoding precursor 28S ribosomal RNA (rRNA), 13,14 which is present as multicopy loci (rDNA) transcribed by RNA polymerase I. 15 R2 is among the most phylogenetically widespread eukaryotic mobile elements but is not present in mammals, 6 which instead have non-site-specific…”

“This value is about 50 times higher than the estimate of approximately 200 units in species of Drosophila [26,27] and 4–12 times larger than estimates in other orthopterans.”

“Ye and Eickbush [7] showed that inserted and un-inserted rDNA units are not different in terms of nuclease sensitivity, psoralen cross-linking, or in epigenetic modifications to their core histone proteins.”

“…it has been estimated that, in certain stocks, approximately 240 rDNA genes may be uninserted and transcriptionally functional and yet, based in bobbed mutations, the total number of active rDNA genes required for normal development and cell function is only around 24 (Ye and Eickbush 2006).”

“For D. melanogaster (Ye and Eickbush, 2006; Eickbush and Eickbush, 2010) and Blattella germanica (Kapelinskaya et al. unpublished data), it was experimentally shown that R2 retrotransposons are transcribed together with 28S rRNA.”

“However, little is described about the transcription level of inserted and uninserted rDNA, or whether there is cotranscription of the rDNA and the retrotransposon (Eickbush and Eickbush 2003; Ye and Eickbush 2006).”

“A large portion of the rDNA copies in a cell are transcriptionally silent, as seen in human cells [172] and other species [173, 174]; differences in rDNA CN do not lead to concomitant changes in rRNA levels [172, 175]; and even artificially silencing copies fails to reduce rRNA expression, since…”

“As expected, several restriction fragments hybridized to the probe and, in agreement with data from photoreacted embryonic chromatin [12], bands representing non-nucleosomal rDNA were not detected in salivary glands.”

“In D. melanogaster , approximately 44% of the rDNA units have R1 retrotransposons inserted at speci fi c locations within their 28S regions 34 .”

“In specific strains of D. melanogaster , as few as 10% of copies are transcriptionally active (Ye and Eickbush 2006).”

“Intriguingly, in contrast to the repressive role of Ars2 in telomeres, it is essential for the transcription of the R1 retrotransposons, which preferentially target rRNA genes and are likely required for their amplification [ 45 ].”

“…rDNA copy number is often in substantial excess of what is required to meet ribosome demands, leading to silencing of the majority of rDNA repeats in a wide variety of species (Conconi et al., 1992, 1989; Dammann et al., 1993; French et al., 2003; McStay and Grummt, 2008; Ye and Eickbush, 2006).”

“This is consistent with studies in Drosophila in which it has been estimated that only 35 to 50 functional rDNA repeats are sufficient for normal development (Ye and Eickbush 2006).”

“It has been shown that rDNA units harboring R1 and R2 insertions are expressed at levels several orders of magnitude lower than intact, actively transcribed rDNA genes, although the exact mechanisms underlying their silencing remain poorly understood [50, 65 – 67].”

“They have been well studied in insects for over 40 years [1–4] R2 elements and another non-LTR retrotransposon, R1 , preferentially insert into the 28S regions of rDNA genes [5].”

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

“However, the transcription level of rDNA with R1 and R2 insertions is much lower than that of rDNA without insertions (Ye and Eickbush, 2006).”

“The expression of these inserted rDNA genes is usually at very low frequencies (Long and Dawid, 1979; Ye and Eickbush, 2006).”