Most Caenorhabditis elegans microRNAs Are Individually Not Essential for Development or Viability

“We performed a broad phenotypic study of all available miRNA loss-of-function mutants, including mutants that had been reported earlier [2,4,20,52].”

“]. Because many messages have been under selective pressure to preserve pairing to a 6mer in the 5′ region of the miRNA known as the miRNA seed (nucleotides 2–7), targets of metazoan miRNAs can be predicted above the background of false-positives by searching for conserved matches to the seed region [”

“In animals, miRNAs are transcribed as long RNA precursors (pri-miRNAs), which are processed in the nucleus by the RNase III enzyme complex Drosha-Pasha/DGCR8 to form the approximately 70-base pre-miRNAs [21–25] or are derived directly from introns [26,27].”

“miRNAs have been identified in organisms as diverse as roundworms, flies, fish, frogs, mammals, flowering plants, mosses, and even viruses, using genetics, molecular cloning, and predictions from bioinformatics [7–16].”

“elegans miRNAs [87,88].”

“We performed a broad phenotypic study of all available miRNA loss-of-function mutants, including mutants that had been reported earlier [2,4,20,52].”

“Because many messages have been under selective pressure to preserve pairing to a 6mer in the 59 region of the miRNA known as the miRNA seed (nucleotides 2–7), targets of metazoan miRNAs can be predicted above the background of false-positives by searching for conserved matches to the seed region [41–45].”

“Because many messages have been under selective pressure to preserve pairing to a 6mer in the 59 region of the miRNA known as the miRNA seed (nucleotides 2–7), targets of metazoan miRNAs can be predicted above the background of false-positives by searching for conserved matches to the seed region [41–45].”

“Knockdown of miRNAs using modified 29 O-methyl antisense oligoribonucleotides also has been reported for the mouse [80].”

“elegans affects vulval development [89,90].”

“In animals, miRNAs are transcribed as long RNA precursors (pri-miRNAs), which are processed in the nucleus by the RNase III enzyme complex Drosha-Pasha/DGCR8 to form the approximately 70-base pre-miRNAs [21–25] or are derived directly from introns [26,27].”

“In animals, miRNAs are transcribed as long RNA precursors (pri-miRNAs), which are processed in the nucleus by the RNase III enzyme complex Drosha-Pasha/DGCR8 to form the approximately 70-base pre-miRNAs [21–25] or are derived directly from introns [26,27].”

“elegans miRNAs were cloned and verified in northern blot experiments [10,11,17,85].”

“Pre-miRNAs are exported from the nucleus by Exportin-5 [28], processed by the RNase III enzyme Dicer, and incorporated into an Argonaute-containing RNA-induced silencing complex (RISC) [29].”

“elegans and humans [94], appears not to have been an exception: for 15 miRNA families, miRNAs with identical seeds have been found in C.”

“Because many messages have been under selective pressure to preserve pairing to a 6mer in the 59 region of the miRNA known as the miRNA seed (nucleotides 2–7), targets of metazoan miRNAs can be predicted above the background of false-positives by searching for conserved matches to the seed region [41–45].”

“For example, the miRNA miR-1 is expressed in muscles of Drosophila, the zebrafish, and the mouse [11,56,98].”

“elegans and mouse development [67,76].”

“In animals, miRNAs are transcribed as long RNA precursors (pri-miRNAs), which are processed in the nucleus by the RNase III enzyme complex Drosha-Pasha/DGCR8 to form the approximately 70-base pre-miRNAs [21–25] or are derived directly from introns [26,27].”

“elegans [66,67], Drosophila [68,69], the zebrafish [70,71], and the mouse [72–75].”

“The first loss-of-function studies of miRNAs in the mouse have been reported demonstrating a role for miR-1 and miR208 in cardiac growth in response to stress [61,62] and miR155/BIC in normal immune function [63,64].”

“The first miRNA studied functionally in Drosophila is encoded by the bantam locus, which had previously been identified in a screen for deregulated tissue growth [54].”

“miRNAs have been identified in organisms as diverse as roundworms, flies, fish, frogs, mammals, flowering plants, mosses, and even viruses, using genetics, molecular cloning, and predictions from bioinformatics [7–16].”

“Binding of the silencing complex causes translational repression [31–33] and/or mRNA destabilization, which is sometimes through direct mRNA cleavage [34,35], but usually through other mechanisms [36–40].”

“elegans [66,67], Drosophila [68,69], the zebrafish [70,71], and the mouse [72–75].”

“For example, the miRNA miR-1 is expressed in muscles of Drosophila, the zebrafish, and the mouse [11,56,98].”

“The first loss-of-function studies of miRNAs in the mouse have been reported demonstrating a role for miR-1 and miR208 in cardiac growth in response to stress [61,62] and miR155/BIC in normal immune function [63,64].”

“For example, the severity of the muscle defect of C. elegans mir-1 mutants might depend on physiological conditions, as is the case for the Dmir-1 mutant phenotypes of Drosophila [56].”

“Binding of the silencing complex causes translational repression [31–33] and/or mRNA destabilization, which is sometimes through direct mRNA cleavage [34,35], but usually through other mechanisms [36–40].”

“elegans [66,67], Drosophila [68,69], the zebrafish [70,71], and the mouse [72–75].”

“In animals, miRNAs are transcribed as long RNA precursors (pri-miRNAs), which are processed in the nucleus by the RNase III enzyme complex Drosha-Pasha/DGCR8 to form the approximately 70-base pre-miRNAs [21–25] or are derived directly from introns [26,27].”

“elegans miRNAs were cloned and verified in northern blot experiments [10,11,17,85].”

“A second Drosophila miRNA, miR-14, also reduces programmed cell death [55].”

“]. Binding of the silencing complex causes translational repression [”

“miRNAs have been identified in organisms as diverse as roundworms, flies, fish, frogs, mammals, flowering plants, mosses, and even viruses, using genetics, molecular cloning, and predictions from bioinformatics [7–16].”

“elegans genome [93], about 60% of miRNAs are members of a family of two to eight genes [19].”

“In animals, miRNAs are transcribed as long RNA precursors (pri-miRNAs), which are processed in the nucleus by the RNase III enzyme complex Drosha-Pasha/DGCR8 to form the approximately 70-base pre-miRNAs [21–25] or are derived directly from introns [26,27].”

“miRNAs have been identified in organisms as diverse as roundworms, flies, fish, frogs, mammals, flowering plants, mosses, and even viruses, using genetics, molecular cloning, and predictions from bioinformatics [7–16].”

“Binding of the silencing complex causes translational repression [31–33] and/or mRNA destabilization, which is sometimes through direct mRNA cleavage [34,35], but usually through other mechanisms [36–40].”

“elegans [66,67], Drosophila [68,69], the zebrafish [70,71], and the mouse [72–75].”

“miRNA function has also been inferred from studies in which miRNAs have been misexpressed in worms, flies, frogs, mice, and cultured mammalian cells [65].”

“Pre-miRNAs are exported from the nucleus by Exportin-5 [28], processed by the RNase III enzyme Dicer, and incorporated into an Argonaute-containing RNA-induced silencing complex (RISC) [29].”

“during studies of the control of developmental timing [”

“We performed a broad phenotypic study of all available miRNA loss-of-function mutants, including mutants that had been reported earlier [2,4,20,52].”

“Binding of the silencing complex causes translational repression [31–33] and/or mRNA destabilization, which is sometimes through direct mRNA cleavage [34,35], but usually through other mechanisms [36–40].”

“The let-7 miRNA controls hypodermal cell-fate decisions during late-larval development by regulating the lin-41, hbl-1, daf-12, and pha-4 mRNAs [48–51].”

“Within the silencing complex, metazoan miRNAs pair to the mRNAs of protein-coding genes, usually through imperfect base-pairing with the 39-UTR, thereby specifying the posttranscriptional repression of these target mRNAs [6,30].”

“For example, the severity of the muscle defect of C. elegans mir-1 mutants might depend on physiological conditions, as is the case for the Dmir-1 mutant phenotypes of Drosophila [56].”

“elegans [66,67], Drosophila [68,69], the zebrafish [70,71], and the mouse [72–75].”

“elegans [78,79] and appears to offer sequence-specific inhibition of small RNAs for a limited time span.”

“elegans miRNAs were cloned and verified in northern blot experiments [10,11,17,85].”

“miRNAs have been identified in organisms as diverse as roundworms, flies, fish, frogs, mammals, flowering plants, mosses, and even viruses, using genetics, molecular cloning, and predictions from bioinformatics [7–16].”

“elegans [78,79] and appears to offer sequence-specific inhibition of small RNAs for a limited time span.”

“elegans [66,67], Drosophila [68,69], the zebrafish [70,71], and the mouse [72–75].”

“elegans [66,67], Drosophila [68,69], the zebrafish [70,71], and the mouse [72–75].”

“We previously described our studies of knockouts of three additional miRNA genes [52], and deletions in two other miRNA genes had been obtained by the C.”

“The muscle-specific Drosophila miRNA miR-1 is required for larval development and cardiac differentiation [56,57].”

“However, the predicted mRNA targets of miRNAs might not share the same degree of conservation as miRNA expression patterns—the spectrum of predicted mRNA targets varies significantly among metazoans [99].”

“elegans [66,67], Drosophila [68,69], the zebrafish [70,71], and the mouse [72–75].”

“Binding of the silencing complex causes translational repression [31–33] and/or mRNA destabilization, which is sometimes through direct mRNA cleavage [34,35], but usually through other mechanisms [36–40].”

“If let-60 RAS is a target of miR-84, loss of mir-84 might result in let-60 RAS overexpression and possibly a multivulva phenotype [91,92].”

“In nematodes, at least 10% of the protein-coding messages appear to be conserved targets of miRNAs [46].”

“Binding of the silencing complex causes translational repression [31–33] and/or mRNA destabilization, which is sometimes through direct mRNA cleavage [34,35], but usually through other mechanisms [36–40].”

“The let-7 miRNA controls hypodermal cell-fate decisions during late-larval development by regulating the lin-41, hbl-1, daf-12, and pha-4 mRNAs [48–51].”

“For example, genetic data indicate that Drosophila mir-7 directly regulates the transcriptional repressor Yan in the fly eye, but that loss ofmir-7 does not appreciably alter eye development, probably because of redundant protein turnover mechanisms that can also downregulate Yan [58].”

“The let-7 miRNA controls hypodermal cell-fate decisions during late-larval development by regulating the lin-41, hbl-1, daf-12, and pha-4 mRNAs [48–51].”

“Injection of individual 29 O-methyl antisense oligoribonucleotides complementary to the 46 miRNAs known to be expressed in the fly embryo resulted in a total of 25 different abnormal phenotypes, including defects in patterning, morphogenesis, and cell survival [77].”

“elegans, flies, fish, and mammals, and several additional families are predicted to be conserved throughout these diverse lineages [19,95–97].”

“(2) Pharyngeal pumping: Frequency of grinder displacement was counted for 20 s by eye, but otherwise as described previously [102] (n 1⁄4 5).”

“Deletion mutants were isolated from a frozen library of worms mutagenized with ethyl methanesulphonate (EMS), 1,2,3,4-diepoxybutane (DEB), or a combination of UV irradiation and thymidine monophosphate (UV-TMP) [82,83].”

“Some miRNAs were predicted based on pre-miRNA folds and verified using northern blotting or PCR with specific primers and cloned miRNA libraries [17,18,85,86].”

“Finally, Drosophila miR-9a is required for sensory organ precursor specification [59], and Drosophila miR-278 is required for energy homeostasis [60].”

“miRNAs have been identified in organisms as diverse as roundworms, flies, fish, frogs, mammals, flowering plants, mosses, and even viruses, using genetics, molecular cloning, and predictions from bioinformatics [7–16].”

“Finally, Drosophila miR-9a is required for sensory organ precursor specification [59], and Drosophila miR-278 is required for energy homeostasis [60].”

“Deletion mutants were isolated from a frozen library of worms mutagenized with ethyl methanesulphonate (EMS), 1,2,3,4-diepoxybutane (DEB), or a combination of UV irradiation and thymidine monophosphate (UV-TMP) [82,83].”

“elegans, flies, fish, and mammals, and several additional families are predicted to be conserved throughout these diverse lineages [19,95–97].”

“the control of developmental timing and likely regulate the hbl-1 mRNA, but act earlier in development than the let-7 miRNA [52,53].”

“org/ [107].”

“We made extensive use of the ‘‘poison’’ primer method, which increases the sensitivity of detection of small deletions [84].”

“Of the 95 C. elegans miRNA genes tested, only a few have grossly abnormal phenotypes when individually knocked out (Miska et al., 2007).”

“…Importance of miRNAs Systematic generation and analysis of miRNA knockouts in C. elegans created the initial impression that most metazoan miRNAs—even most miRNAs that are broadly conserved— have only subtle or redundant biological functions (Miska et al., 2007; Alvarez-Saavedra and Horvitz, 2010).”

“elegans coupled with in-depth screens for altered morphology, growth, development, and behavior identified only four gene knockouts with detectable phenotypes (Miska et al., 2007).”

“and Human Disease”

“In worms, most individual miRNA mutants show no gross phenotype (Miska et al., 2007); the same is true for several of the mouse knockouts generated to date, including miR-21, miR-210, miR-214, miR-206, and miR-143 (E. Olson, personal communication).”

“elegans strains that carry mutations of multiple miRNA-encoding loci suggest that this is indeed the case in certain instance”

“Perhaps somewhat surprisingly, systematic inactivation of individual miRNA in flies and worms has revealed that many miRNAs are dispensable for development or viability under standard laboratory conditions (Bushati and Cohen, 2007; Leaman et al., 2005; Miska et al., 2007).”

“In the worm, let-7 is a component of the heterochronic pathway; it is expressed at a late stage of larva development, regulates the transition from larva to adult(5) and is embryonic lethal.(140) In the fruit fly, let-7-knockout flies are externally normal but exhibit behavioral defects and juvenile features in their…”

“This canalizing function of miRNAs can explain why a loss of an miRNA often does not result in a visible phenotyp”

“elegans show that single and even double and triple mutants do not result in easily recognizable phenotype”

“elegans and Drosophila, and has also been reported for many individual miRNAs in the mouse [8,10,65-71].”

“It also explains why miRNAs frequently appear dispensable under uniform laboratory conditions (Bushati and Cohen, 2007; Leaman et al., 2005; Miska et al., 2007).”

“Furthermore, these analyses may also be complicated by functional redundancy of important regulatory molecules, even for conserved miRNAs with roles in development, which individually are mostly not essential for C. elegans viability (Miska et al. 2007).”

“New deletion alleles for prg-1 and prg-2 were generated as described previously (Miska et al., 2007) and are listed in Table 3.”

“Additionally, gene knockout studies in animals demonstrate that numerous miRNAs are not essential for viability [30,31].”

“One fifth of these lines have obvious morphological defects, which is in the same range as the approximately 10% of miRNA knockouts that caused phenotypic abnormalities or lethality in Caenorhabditis elegans [31].”

“Jo ur na l o f C el l S ci en ce these miRNAs do not affect the developmental progression of C. elegans (Miska et al., 2007). mir-71, -238 and -246 mutants display a significantly shorter lifespan than those of wild-type animals, and overexpressing miR-71 or miR-246 increases lifespan, indicating…”

“Just over 40% of these genes have been deleted, and most do not display an overt phenotype (Miska et al. 2007).”

“let-7 family animals were scored after passage through dauer to suppress the early adult lethality [1].”

“However, none of these mutants exhibited any physiological or behavioral abnormalities during growth and development [25, 26]; Figure S4 and Table S4).”

“elegans can tolerate removal of most individual miRNAs without obvious developmental defects.((40)) The lack of clear miRNA hubs and the fact that most of them are not essential for viability both agree with the hypothesis that miRNAs do not function as master regulators, but rather predominantly function to fine tune…”

“These preliminary considerations suggest that if the primary function served by miRNA gene regulation is to stabilize gene expression levels, miRNA knockouts may not have strongly deleterious phenotypes in individuals,((45)) as recently realized in a large-scale knockout study.((78)) Although we are in very early days…”

“…with disease, cancer, or phenotypic abnormalities (Bartel 2004, 2009; He and Hannon 2004; Sokol and Ambros 2005; Calin and Croce 2006; Esquela-Kerscher and Slack 2006; Li et al. 2006; Ghildiyal and Zamore 2009), yet miRNA knockouts often result in only subtle phenotypic effects (Miska et al. 2007).”

“This is the first demonstration that mir-34 plays a role in the response to radiation in vivo and is the first known phenotype associated with mir-34 deletion mutants in C. elegans (Miska et al., 2007) or in any animals.”

“It has proven difficult to determine the importance of isomiRs both because of the limited tools that are available for modulating the levels of specific isomiRs (16) and because of the overlapping and redundant nature of most miRNA genes (24).”

“In worms, ,10% of systematic miRNA deletions cause embryonic death or grossly abnormal phenotypes (90).”

“Beyond differences in lifespan and stress-resistance [36], mir-71 mutant animals appear phenotypically wild-type [46].”

“First, we assayed the regulation of the wild-type cog-1 39 UTR sensor in mir265 null mutants (Miska et al. 2007) and found no effect on sensor gene expression (Fig.”

“elegans have provided crucial insights into miRNA function; not all miRNAs that change with age lead to modulated lifespan upon altered activity [90], and many miRNAs are not required for development or viability [91, 92].”

“elegans, where less than 10% of miRNAs give rise to embryonic developmental or grossly abnormal phenotypes but which may appear in sensitized backgrounds or under stress (Alvarez-Saavedra and Horvitz, 2010; Brenner et al., 2010; Miska et al., 2007).”

“Initial studies support that evaluation of functional redundancies will be an important factor in this analysis [39,40,42,48] and that conserved regulatory functions may shed light on disease mechanisms [6,42].”

“Remarkably, although loss-of-function mutation of most individual miRNAs yields no overt developmental defects in multiple organisms (Miska et al., 2007), the essential role of miR-219 in myelination indicates that miR-219 plays a central role in rather than just fine-tuning myelinogenesis.”

“…miRNAs or miRNA seed families in C. elegans leads to developmental phenotypes including death of embryos or early larvae and defects in locomotion, body size, egg laying, and dauer larvae formation (Miska et al. 2007; Alvarez- Saavedra and Horvitz 2010; Brenner et al. 2010; Shaw et al. 2010).”

“Future studies may reveal a phenotype in organ homeostasis over time or under stress and pathological conditions, as was revealed for many microRNA knockouts [54, 55].”

“S7 and Supplementary Table S2): (1) unregulated transcription, that is, the mRNA is transcribed from a constitutive promoter30; (2) regulated transcription where the protein activates its own transcription (that is, the PFL motif); (3) a NFL mediated by the miRNA inducing degradation of the target mRNA (that is, a NFL…”

“Some sets of miRNA map to specific chromosomal clusters, as in the case of miR-35 to miR-41, which have redundant functions in embryonic development [42] and are abundantly expressed in the embryonic stage (Figures 3b and 7).”

“able phenotypes have been identified in MIRs in plants and other organisms (Miska et al., 2007; Voinnet, 2009).”

“Studies with this collection suggested that the majority of miRNA mutants had no detectable phenotypes when mutated individually (Miska et al., 2007).”

“However, genetic KO models in either C. elegans or mouse have implicated very few individual miRNAs as indispensable for animal development 14, 15, 42 .”

“…the most ancient animal miRNA, mir-100 (Grimson et al., 2008); let-7 is also highly conserved and plays broad and essential roles in many contexts (Mondol and Pasquinelli, 2012); and the mir-430/302/290 family also seems to have maintained a core position in development during vertebrate…”

“However, because many miRNAs can be deleted without resulting in any apparent phenotype, there is also a growing consensus that most miRNAs do not play an indispensable role in dictating the outcomes of biological responses (Miska et al., 2007).”

“elegans, the majority of individual miRNAs are not required for viability or for development, as most loss of function miRNA mutants display no obvious developmental abnormalities [1].”

“elegans are not individually essential for viability under standard laboratory conditions, the possibility that they fulfill conditional functions such as stress response is particularly appealing [77].”

“In fact, most individual miRNAs (52) and even many miRNA families altogether (53) are dispensable under homeostatic conditions, indicating that a certain degree of fluctuation or imprecision of gene expression is well tolerated.”

“Although a study in which 95 miRNA genes were mutated in Caenorhabiditis elegans, no abnormal phenotypes were observed, indicating major miRNAs are not vital for the development or viability.(7) Increasing evidence supports the idea that miRNAs participate in complex regulatory networks and the abnormality of some miR…”

“However, genetic knockout of specific miRNAs renders the mutant animals incapable of coping with stress (Miska et al , 2007; Alvarez-Saavedra & Horvitz, 2010).”

“Experiments in simple organisms that show most miRNAs are not essential, and individually often have subtle effects on their downstream targets, provide evidence for the theory of miRNA redundancy.(14,15) Five of 7 of our miRNAs are members of the same gene family (miR-200).”

“Together, these results confirm attenuation of IEG mRNA abundance by microRNAs and are consistent with studies indicating that promoters of IEGs are poised for activation in resting cells (23, 24). miR-370 miR-188 miR-671 miR-320 miR-198 miR-134 miR-662 miR-575 miR-212 miR-564 miR-638 miR-572 miR-202 miR-452 miR-630 m…”

“elegans miRNAs are knocked out individually or in combination as a whole family that shares the same seed sequence, only a few exhibit developmental defects (Alvarez-Saavedra and Horvitz, 2010; Miska et al., 2007).”

“elegans strains with mutations for aging-associated miRNAs have been characterized in aging,(41,67) made possible by a large collection of miRNA loss-of-function mutants.(68) When characterized during development, most of these strains did not demonstrate gross abnormalities(68); nevertheless, mutations for some aging…”

“A genome-wide miRNA knockout resource for the functional analysis of miRNAs currently exists only for the invertebrate Caenorhabditis elegans”

“As previously described, mir-124 mutants are viable with no gross abnormalities (77).”

“Knock-out studies in model organisms have had only limited success in delineating miRNA function, possibly because redundant miRNAs exist at saturating levels in wild-type cells, or because of compensatory effects in downstream signaling pathways (8).”

“elegans , loss of most individual miRNA genes caused no obvious phenotyp”

“Genetic knockouts and mutations for 95 miRNAs have been publishe”

“Many miRNAs have subtle if not undetectable loss-of-function phenotypes (Miska et al., 2007; Alvarez-Saavedra and Horvitz, 2010), but it is also the case that many miRNA mutants produce synthetic phenotypes in combination with other genetic insults (Brenner et al.”

“Systematic mutagenesis of many individual miRNA genes (Miska et al., 2007) and paralogous families of miRNA genes (Alvarez-Saavedra and Horvitz, 2010) were performed in the nematode Caenorhabditis elegans.”

“Following the studies by Horvitz and colleagues (7, 8) that showed that single mutations of the vast majority of miRNAs did not reveal obvious developmental or growth defects, our study reinforces the idea that most miRNAs do not regulate specific physiological functions through a robust regulatory interaction between…”

“For example, deletions of each miRNA gene individually in Caenorhabditis elegans results in few, if any, phenotypic consequences [49].”

“miRNAs may act redundantly [42] or in concert [43,44].”

“elegans mir deletion strains [20] for differences in autofluorescent age pigment levels in old animals.”

“elegans miRNAs, either individually [23] or by combined mutation of related sequences [24], results in no observable effect on development or viability.”