Identification of over 15,000 unique 21U-RNA species in C. elegans
We used Solexa sequencing technology (Seo et al., 2004
) to generate 29,112,356 small-RNA cDNA reads that perfectly matched the C. elegans
genome. Among these we identified 971,981 reads from 15,458 unique loci with properties similar to previously defined 21U-RNA loci (Ruby et al., 2006
). These new reads matched 95.1% of the 5,454 previously sequenced 21U-RNAs and 78.3% of the 10,644 previously predicted 21URNAs (Ruby et al., 2006
) and brought the total number of unique experimentally confirmed 21U-RNA loci to 15,722.
A common characteristic of 21U-RNA loci is the presence of an upstream sequence motif (, (Ruby et al., 2006
)). As previously observed, RNA species 21nts in length could be separated into two distinct sets based on the motif scores of their genomic loci (). Species with a high motif score also tended to exhibit the other essential features, including 21nt length and 5′ U nucleotide, that together define the 21U-RNA class (Supplemental Figure 1 A-C
21U-RNAs can be distinguished from other RNA species by their lengths and upstream motif matches
21U-RNAs with strong upstream motif matches were concentrated in two broad regions along chromosome IV ( and Ruby et al., 2006
). Supporting the potential importance of this motif in 21U-RNA biogenesis, the motif score strongly correlated with the magnitude of 21U-RNA, as indicated by the number of sequenced reads in our data sets (). Despite the presence of many high-scoring 21U-RNA motifs in orthologous regions of the C. briggsae
genome, the 21U-RNA sequences themselves were not conserved. Even in rare cases in which the core of the upstream motif was perfectly aligned to a high-scoring motif within a syntenic region of the C.
genome (Blanchette et al., 2004
), the sequence of the consequent 21U-RNA was essentially nonconserved (). Only approximately 6% of the 21U-RNA loci and/or motifs were unambiguously aligned within syntenic regions in C. briggsae.
In these few cases, this was often due to overlap with annotated coding exons, which rarely contain 21U-RNAs (Supplemental Figure 1D
). The only portion of the 21U-RNA flanking regions with elevated conservation frequencies above background was the 8nt core of the upstream motif (Supplemental Figure 1E
21U-RNAs are expressed in the C. elegans germ line
The developmental dynamics of 21U-RNA expression were examined by Northern blot analysis using probes specific for 21U-RNA-1 and 21U-RNA-3442. Both small RNAs were expressed at low levels from the L1 to L3 stage, began to accumulate to high levels during the L4 stage, and reached maximal expression in the young-adult and gravid-adult stages (). This pattern of expression correlated with the proliferation of the germ line, and was consistent with a germ-line origin. Both RNAs were expressed at approximately equal levels in male- or female- enriched populations (), but were absent in RNA samples prepared from germ-line-deficient glp-4(bn2) and eft-3(q145) mutant populations (). Finally, both small RNAs were present in embryos (), which may reflect maternal and/or paternal loading.
21U-RNAs are expressed in the C. elegans germ line
High throughput sequencing indicated that the developmental expression profile for the entire class of 21U-RNAs, was identical to that of 21U-RNA-1 and 21U-RNA-3442 (). The number of sequenced reads for each 21U-RNA species increased dramatically in late larval and adult stages. Furthermore, the number of reads was reduced (130 fold), from 5.8% to just 0.04% of total reads, in animals lacking a germline (). Adult hermaphrodites switch to an exclusively female mode of gametogenesis and store only 200−300 mature sperm. The relative abundance of various individual 21U-RNA species was comparable between male and adult hermaphrodite populations, suggesting that very similar 21U-RNA populations are present in germlines undergoing to oogenesis and spermatogensis.
PRG-1 is expressed in the germ line and required for 21U-RNA accumulation
To examine whether the accumulation of 21U-RNA-1 and 21U-RNA-3442 was dependent on known components of the RNAi machinery, we systematically examined RNA prepared from mutant strains lacking specific components of the RNAi pathway. The accumulation of 21U-RNAs did not require the wild-type activities of any of the previously described RNAi pathway components, including DCR-1 ( Left and Supplemental Figure 2
PRG-1 protein is expressed in the germ line and required for 21U-RNA accumulation
To determine if accumulation of 21U-RNAs is dependent of any AGO proteins, we also analyzed mutant strains representing all of the C. elegans AGO family members, including several multiple-mutant strains. Only prg-1 mutants lacked 21U-RNA-1 and 21U-RNA-3442 ( Right and data not shown). Strains mutant for prg-2, a nearly identical homolog of prg-1, did not exhibit defects in 21U-RNA expression ( Right). We observed no defects in miRNA expression or in the expression of any of several other species of endogenous siRNA examined in these mutants (data not shown). Moreover, prg-1 mutants exhibited a wild-type RNAi response to foreign dsRNA (data not shown). These findings suggested that prg-1 was defective specifically in the 21URNA pathway.
Consistent with the genetic requirement of prg-1 for 21U-RNA accumulation, the stage-specific expression of PRG-1 protein was coincident with that of 21U-RNA-1 and 21U-RNA-3442. PRG-1 levels were reduced in L1/L2 and L2/L3 worms when compared with L4 worms, as well as young and gravid adults (). As observed for 21URNAs, we could also detect the PRG-1 protein in embryo extracts, and we were unable to detect PRG-1 in the glp-4(bn2) mutant strain, suggesting that this protein is expressed in the germline. PRG-1 was also present in protein extracts from both female- and male- enriched populations. Curiously, the expression of prg-1 was reduced in wild type worms cultured at 25°C (). Analysis of the expression of the prg-1/prg-2 mRNA by real-time PCR revealed an expression pattern similar to that observed for the PRG-1 protein. The only exception observed was in the embryonic stage (). Although we could detect a high level of the PRG-1 protein in embryos, the mRNA was almost undetectable, supporting the idea that PRG-1 complexes in embryos are parentally derived.
In wild type worms, we observed a striking localization of PRG-1 in the cytoplasm and in prominent cytoplasmic structures in germ cells at nearly all stages of germ-line development. In both hermaphrodites and males, PRG-1 formed perinuclear foci in both the mitotic and meiotic zones of the germ line (). In mature oocytes the staining persisted but PRG-1 foci lost their perinuclear association and became dispersed in the cytoplasm ( and data not shown). In males all PRG-1 staining disappeared abruptly as spermatids matured (). The pattern of PRG-1 localization, including its localization during embryogenesis (), resembled that of P granules, which are components of the C. elegans
germ-line cytoplasm, or nuage (Strome and Wood, 1982
) (Strome, 2005
). Indeed, the localization of PRG-1 perfectly overlapped throughout development the localization of the previously described P-granule component, PGL-1 (Kawasaki et al., 1998
21U-RNAs depend on and interact physically with PRG-1
To determine whether PRG-1 is required more broadly for 21U-RNA accumulation, we performed high-throughput sequencing analysis on small-RNA populations prepared from prg-1 mutant animals and from wild-type animals reared at 20°C. For wild-type animals, approximately 11% of the 1,789,450 genome-matching reads corresponded to the 21U-RNAs, whereas for prg-1 mutant animals less than 0.05% of the 1,774,442 genome-matching reads corresponded to 21U-RNAs (). This dramatic reduction in 21U-RNAs resembled that observed in animals lacking a germ line altogether (). However, prg-1 animals maintained at 20°C were fertile and exhibited nearly wild-type levels of another class of germ-line enriched small RNAs, the endogenous siRNAs (). These findings indicate that prg-1 is required for the accumulation of the entire 21U-RNA class of small RNAs.
PRG-1 interacts with and is required for the accumulation of all 21URNAs
To examine whether the 21U-RNAs physically interact with PRG-1, we immunoprecipitated the PRG-1 protein complex along with associated RNA. Both 21URNA-1 and 21U-RNA-3442 co-precipitated with the PRG-1 immune complex but not with precipitates recovered using pre-immune serum (). Small RNA species that did not require PRG-1 activity for accumulation, such as miR-66, were not detected in PRG-1 immunoprecipitates (). In contrast, we found that ALG-1/ALG-2 AGO-associated immune complex contained miR-66 but not 21U-RNA-1 or 21U-RNA-3442 ().
Biochemical analysis of small RNAs recovered in the PRG-1 IP complex demonstrated a strong bias for small RNAs with 5′ U (>91%) compared to the total input population, which was enriched for 5′ G (>70%; ). Similarly, deep sequencing of small RNA libraries prepared from the IP sample demonstrated a dramatic enrichment for 21nt RNAs with 5′ U in the PRG-1 complex (). In addition, 21mers with high-scoring motif matches were dramatically enriched in the IP sample (), and mapped comprehensively across the previously described 21U-RNA clusters on chromosome IV (). No other RNA species was significantly enriched in the PRG-1 IP. The above observations suggest that PRG-1 specifically binds 21U-RNAs to form a complex important for germ-line function and fertility.
prg-1 mutants exhibit a broad spectrum of germ-line defects
A previous study demonstrated that RNAi targeting prg-1
leads to reduced fertility (Cox et al., 1998
). In this previous study it was not possible to target these two genes separately because of their high level of sequence identity. However, our examination of the phenotypic contributions of recently identified probable null alleles, revealed that most, if not all, of the germ-line defects result from the absence of prg-1
. For example, prg-2
mutants exhibited wild-type brood sizes at both 20°C and 25°C () as well as normal numbers of morphologically wild-type germ cells (compare ). In contrast, prg-1
mutants exhibited dramatically reduced fertility at both temperatures (). Consistent with this phenotype, two different prg-1
mutant strains and a prg-1 prg-2
double-mutant strain all exhibited a significant reduction in the total number of germ nuclei populating the adult gonad (). The numbers of germ nuclei were reduced in each zone, but were most dramatically reduced in the mitotic zone in these mutants. The reduction in germ cell numbers was observed at all temperatures, and thus does not by itself explain the sterility of prg-1
mutants at 25°C.
PRG-1 exhibits a broad spectrum of germ line defects
mutants exhibit temperature-dependent sterility, they do not appear to encode thermo-labile products. Rather, both alleles examined in this study are likely to represent null mutations (Cuppen et al., 2007
) (Supplemental Figure 3A
). As expected for null-mutants, the PRG-1 protein was either absent or truncated in these mutant strains at all temperatures (Supplemental Figure 3B
). Furthermore, the 21U-RNA depletion associated with prg-1
mutants was observed at all temperatures examined, including the semi-permissive temperatures of 15°C and 20°C. These findings suggest that in addition to their role in maintaining proper germ-cell numbers at all temperatures, PRG-/21U-RNA complexes may function at higher temperatures to facilitate an otherwise temperature-dependent germ-line process required for normal fertility.
Temperature-shift experiments demonstrated that the temperature-sensitive period of prg-1 mutants occurs during the adult stage. The fertility of animals shifted down from 25°C as young adults was substantially rescued, to an average of 40 progeny (n=10). Conversely, maintaining animals at 15°C during the L1 to adult stage, when the germ line is proliferating most rapidly, did not significantly rescue the fertility defect. These results suggest that, the germ cells produced in prg-1 null mutant animals (that entirely lack PRG-1 protein expression), are deficient in a process important for their functionality (ovulation or fertilization) at elevated temperature.
To examine the relative contribution of defects in sperm vs oocytes to the reduced fertility of prg-1 mutants, mutant hermaphrodites raised at 25°C were mated to wild-type males. The temperature-dependent sterility of prg-1 was partially rescued, as the average number of prg-1 progeny produced by animals reared at 25°C was 3 (n=10), but this number increased to 19 (n=10) when prg-1 mutants were mated with wild type males. These findings suggested that the fertility defects of prg-1 hermaphrodites stem, in part, from defects in the production and/or functionality of both the male and female gametes.
In summary, prg-1 mutants exhibit dramatically reduced germ-cell numbers at all temperatures, and the gametes produced are markedly more sensitive to temperature than are those of wild-type animals. For example, at 25°C, wild-type animals produce ~200 progeny, about two thirds of the brood size observed at 20°C, while prg-1 mutants produce an average brood size of only 3 progeny at 25°C, less than one tenth the brood size of 40 observed at 20°C. This reduction in brood size at higher temperature is approximately correlated with the reduction in the number of embryos observed, consistent with the idea that ovulation or fertilization are impaired at higher temperature.
prg-1 mutants exhibit surprisingly subtle changes in gene expression
On Chromosome IV hundreds of protein-encoding genes are interspersed with intergenic and intronic 21U-RNA loci over genomic regions that are millions of base pairs in length. Therefore, tiling arrays were used to profile changes in gene expression to determine whether the absence of 21U-RNAs in prg-1
mutants might cause significant perturbations of gene expression either on this autosome or elsewhere. We found that prg-1
and wild-type animals have broadly similar patterns of gene expression. Notably, genes located near 21U-RNA loci, including genes located within and around the major clusters of 21U-RNA loci on Chromosome IV, were not significantly altered in their expression (). Among 88 groups of developmentally co-regulated genes, also referred to as gene ’mountains’ (Kim et al., 2001
), 66 were essentially unchanged between the wild-type and prg-1
strains (). Among the 16 mountains with decreased expression in prg-1
mutants, were several mountains with germ-line functions such as cell division and oogenesis. Among the 6 mountains with increased expression was one containing spermatogenesis-related genes.
prg-1 mutants exhibit surprisingly subtle changes in gene expression
In C. elegans
, a large class of RdRP-derived endogenous siRNAs (endo-siRNAs) target transposons and repetitive sequences as well as numerous protein-encoding genes (Ambros et al., 2003
; Ruby et al., 2006
; Gu and Conte, in preparation). Although PRG-1 does not appear to interact directly with small RNAs of this type ( and Supplemental Table 2
), we wondered whether 21U-RNAs might be linked, perhaps indirectly, to changes in the patterns of endo-siRNA expression. In many instances, changes in endo-siRNA levels correlated inversely with changes in gene expression from the corresponding interval ( and Supplemental Table 4
). However, the regions with significant changes in endo-siRNA levels were not correlated with regions containing 21U-RNAs or sequences with extended sequence similarity to 21U-RNAs.
One curious exception to this finding was the transposon Tc3, within which resides a single 21U-RNA. Found in all 22 Tc3 genomic loci, 21U-RNA-15703 overlaps the 3′ inverted repeat (IR) downstream of and in the same orientation as the transposase gene (). This sequence was identified three times among 2 million reads in our small-RNA library prepared from the PRG-1 immune complex, an apparent enrichment when compared to only 12 reads in over thirty million from the remaining non-IP-associated data set. Examination of the endo-siRNA profile across a representative Tc3 element revealed two types of endo-siRNA reads. The first were antisense to the transposase gene and were unaffected in prg-1(tm872) mutants (). The second were directed, with a marked strand asymmetry, toward the Tc3 IR regions and were severely depleted in prg-1(tm872) mutants (). Neither the IR-directed, nor the transposase-directed siRNAs exhibited co-immunoprecipitation with PRG-1 (). Although the numbers of endo-siRNAs targeting the transposase gene were not significantly reduced in prg-1, we nevertheless observed a 3- to 4-fold up-regulation of the Tc3 transposase mRNA (). Up regulation of the transposon mRNA as well as a greater than 100-fold increase in Tc3 transposition frequency were also observed for two different prg-1 mutant alleles in a parallel study (Das et al.,: See Discussion).