John Bracht ¹, Shuan Hunter ¹, Rachel Eachus, Phillip Weeks and Amy E. Pasquinelli

Department of Biology, University of California, San Diego, La Jolla, California 92093, USA
¹ These authors contributed equally.

Reprint requests to: Amy E. Pasquinelli, Department of Biology, University of California, San Diego, La Jolla, CA 92093, USA;
e-mail: apasquin@ucsd.edu        fax: (858) 822-3021

Members of the microRNA (miRNA) class of 22-nucleotide RNAs regulate the expression of target genes that contain sequences of antisense complementarity. Maturation of miRNAs involves cleavage of longer primary transcripts, but little is yet understood about how miRNA genes are transcribed and enter the processing pathway. We find that relatively long, polyadenylated transcripts encoded by the Caenorhabditis elegans let-7 gene undergo trans-splicing to the spliced leader 1 (SL1) RNA. Deletions, including removal of the trans-splice site, upstream of mature let-7 sequence result in stable accumulation of primary transcripts and compromised production of mature let-7 RNA in vivo. Our data show that multiple steps of let-7 miRNA biogenesis can be uncoupled, allowing for complex regulation in the production of a functional miRNA. Finally, the observation that let-7 primary transcripts undergo splicing highlights the importance of identifying the sequence of endogenous pri-miRNA substrates recognized by the cellular processing machinery. 

The newly established class of regulatory RNAs, called microRNAs (miRNAs), is comprised of ~22-nucleotide RNAs that control expression of target genes via antisense base-pairing (Lai 2003; Bartel 2004). All multicellular organisms are believed to express miRNAs and the Caenorhabditis elegans, Drosophila, and human genomes each have been predicted to encode more than 100 different miRNAs (Grad et al. 2003; Lai et al. 2003; Lim et al. 2003a,b). Already members of this new class of tiny regulatory RNAs have been implicated in diverse biological roles, including temporal patterning, cell death regulation, and control of fat metabolism (Ambros 2003; Bartel 2004).

The founding members of the microRNA gene family, lin-4 and let-7, were identified as essential regulators of temporal development in the nematode C. elegans (Pasquinelli and Ruvkun 2002). The developmental function of lin-4 and let-7 RNAs is related to the timing of their expression: Mature lin-4 RNA first appears midway through the first larval stage to regulate early larval cell divisions (Lee et al. 1993; Feinbaum and Ambros 1999), and mature let-7 RNA is undetectable until the last larval stages when it is needed to direct larval to adult cellular transitions (Reinhart et al. 2000). The let-7 RNA sequence, its general temporal expression pattern, proteins involved in its maturation, and genes controlled by this small RNA are conserved from nematodes to humans (Pasquinelli et al. 2000; Grishok et al. 2001; Hutvagner et al. 2001; Ketting et al. 2001).

The Dicer RNase processes let-7, and likely all other miRNA precursors (pre-miRNAs), to the mature ~22-nt forms (Grishok et al. 2001; Hutvagner et al. 2001; Ketting et al. 2001; Lee et al. 2003; Lund et al. 2004). Members of the extensive and highly conserved PAZ (Piwi, Argonaute, Zwille) /Piwi Domain (PPD) protein family (Cerutti et al. 2000; Schwarz and Zamore 2002) also function in the maturation of miRNAs (Grishok et al. 2001; Hutvagner and Zamore 2002; Mourelatos et al. 2002). The nuclear export receptor Exportin-5 is responsible for delivering the ~65-nt stem-loop structured pre-miRNAs to the cytoplasm for this maturation step (Yi et al. 2003; Bohnsack et al. 2004; Lund et al. 2004). Longer primary transcripts, called pri-miRNAs, serve as the initial substrates for processing by the nuclear Drosha RNase to generate the miRNA precursor forms (Lee et al. 2002; Basyuk et al. 2003; Lee et al. 2003; Zeng and Cullen 2003). Although sequences outside of the pre-miRNA hairpin have been shown to be important for RNA processing or stability (Lee et al. 2003; Zeng and Cullen 2003; Chen et al. 2004), little is yet known about the actual composition of pri-miRNAs.

Hundreds of miRNAs have now been isolated from diverse species, but the genomic codes that signal their production are yet to be defined. Some miRNA sequences are embedded in protein coding genes and, thus, may rely on the host gene regulatory elements for transcription (Aravin et al. 2003; Lagos-Quintana et al. 2003; Lai et al. 2003; Lim et al. 2003b). For many miRNA genes, though, there is no evidence for coexpression with messenger RNAs (mRNAs) and, thus, transcription is predicted to be directed by independent promoter elements. Regulatory motifs in miRNA genes that specify transcription by a particular polymerase have remained, for the most part, elusive. There are numerous examples of temporally or spatially restricted miRNAs, which is suggestive of Pol-II-directed transcriptional regulation. The temporal regulatory element (TRE) in the C. elegans let-7 gene can direct developmentally controlled expression of a protein-coding gene (Johnson et al. 2003), implicating Pol-II-mediated synthesis from this promoter. Nonetheless, Pol III promoters have been used to produce functional miRNAs ectopically (Zeng and Cullen 2003; Chen et al. 2004). Determination of the natural transcriptional start sites may facilitate the identification of important elements controlling expression of miRNA genes.

In this article we report the identification of the complete primary transcripts for the let-7 miRNA in C. elegans. We define two polyadenylated transcripts that likely serve as substrates for a trans-splicing reaction that appends Spliced Leader 1 (SL1) sequence to the 5' end of the let-7 pri-miRNA. We present evidence that trans-splicing is important for generation of mature let-7 RNA and that the SL1-appended form of let-7 is a substrate for processing by a C. elegans Drosha homolog. In contrast, factors involved in the terminal steps of let-7 maturation and stabilization, the dicer (dcr-1) and argonaute (alg-1) homologs, do not influence the transcription or initial processing events. These data identify cis-acting sequences and trans-acting factors important for specific steps of let-7 miRNA biogenesis.

Results:

The let-7 miRNA gene encodes long, polyadenylated primary transcripts

In C. elegans, ~2460 nt of genomic DNA sequence containing let-7 is sufficient to fully rescue the let-7(mn112) putative null mutant of this gene (Reinhart et al. 2000). Prior to this study, the 65-nt precursor and 22-nt mature RNAs were the only transcripts detected from the let-7 gene. The let-7 pri-miRNAs may be rapidly processed and difficult to detect by standard Northern analyses. Thus, we employed reverse transcription polymerase chain reaction (RT-PCR) methods to identify potential let-7pri-miRNAs. Using a let-7-specific primer in the first strand RT reaction followed by PCR with forward and reverse primers corresponding to this miRNA gene, we detected the expected size product from wild-type adult but not egg-stage RNA preparations (Fig. 1A, lanes 3,4). No product was amplified in controls where the reverse transcriptase was omitted, verifying that the PCR product is dependent on first-strand synthesis of the RNA template (Fig. 1A, lanes 6–8). Interestingly, RT-PCR reactions using adult stage RNA from let-7(mn112) mutants, which harbor a 193-nt deletion upstream and including the first nucleotide of mature let-7 RNA, produced the expected size band at an apparently higher level relative to that produced from wild-type RNA (Fig. 1A, cf. lanes 4 and 5).

Identification of let-7 primary transcripts

Figure 1. Identification of let-7 primary transcripts.

(A) Total RNA samples from egg and adult stage wild-type worms and adult let-7(mn112) were subjected to reverse transcription (+RT, lanes 3–5) or mock (-RT, lanes 6–8) reactions using A62 primer (see D), followed by PCR with A63 + A62 primers. The products were separated by electrophoresis in a 1% agarose gel and visualized by ethidium bromide staining. PCR of wild-type genomic DNA (lane 2) served as a size marker and specificity control for the PCR reactions. Lanes 1 and 9 show the migration of bands in a 1-kb DNA marker (NEB).

(B) RT-PCR was performed as in A except that oligo dT primer was used for RT, and let-7-specific primers (A63 + A76) and primers for eft-2 mRNA were used in the PCR.

(C) Three DNA products from 5' RACE and one from 3' RACE, indicated by arrowheads, were specifically amplified in reactions containing template from wild-type worms.

(D) The 2460-nt C. elegans genomic fragment of DNA encoding the let-7 transcripts. The deletions immediately upstream of the ~65-nt let-7 RNA precursor in let-7(mg279) and let-7(mn112) mutant strains are depicted with brackets. The positions and names of primers used in this study are indicated with arrows.

Based on our finding that let-7 transcripts contain poly(A) tails, we predicted that these RNAs might also have 5' cap structures that would facilitate identification of the 5' and 3' termini by standard rapid amplification of cDNA ends (RACE) methods. From wild-type RNA samples, three 5' and one 3' RACE products were readily detected (Fig. 1C, lanes 2,7). Cloning and sequencing of these products revealed a common 3' end approximately 70 nt downstream from the poly(A) signal that likely represents the cleavage site (Fig. 1D). The 5' RACE revealed transcripts of ~1731 nt and ~890 nt that initiate 223 nt and 1064 nt downstream from the temporal regulatory element (TRE; Johnson et al. 2003). We also identified a 728-nt transcript that terminates precisely 38 nt upstream of the let-7 precursor sequence and is appended to 22 nt from the 5' end of the spliced leader 1 (SL1) RNA (Fig. 1D). The sequences and positions relative to mature let-7 RNA of the 3' splice consensus sequence (TTTTCAG|G) recognized for trans-splicing by SL1 and the polyadenylation site are conserved in the Caenorhabditis briggsae let-7 gene.

Trans-splicing is important for generation of mature let-7 RNA

Deletions upstream of mature let-7 RNA sequence were originally predicted to interfere with transcription of the RNA gene because less 22-nt let-7 RNA is observed in such mutant backgrounds (Reinhart et al. 2000). The let-7(mg279) (27-nt deletion) or let-7(mn112) (193-nt deletion; see Fig. 1D) mutant strains produce several-fold less or undetectable levels of mature 22-nt let-7 RNA, respectively (Fig. 2A). In contrast, significantly increased levels of longer let-7 transcripts are observed in these deletion mutants compared to wild type, as detected by Northern blot analyses (Fig. 2B). Likewise, RT-PCR experiments using forward primers to detect only the longest transcript (Fig. 2C, first panel) or both the ~1731-nt and ~890-nt transcripts (Fig. 2C, second panel) indicate increased levels of these RNA species in the deletion mutants relative to that observed in wild-type worms.

Expression of mature let-7 RNA correlates with production of SL1-spliced let-7 RNA

Figure 2. Expression of mature let-7 RNA correlates with production of SL1-spliced let-7 RNA.

(A) Total RNA from adult stage wild-type, let-7(mg279) and let-7(mn112) worms was isolated, separated by 11% denaturing PAGE, and probed for precursor and mature let-7 RNA levels by Northern analyses. 5S rRNA detected by ethidium bromide staining of the gel serves as a control for the loading and quality of the RNA samples.

(B) The same RNA samples as A were separated under denaturing conditions in a 1.2% agarose gel and probed for longer let-7 transcripts. Positions of the 3500-nt 28S, 1750-nt 18S, and 152-nt 5.8S rRNAs are indicated. The 28S rRNA detected by ethidium bromide staining is shown as a loading control. Note that the let-7(mg279) sample appears underloaded but, nonetheless, the level of long let-7 transcripts detected in this mutant still greatly exceeds that detected in wild-type worms. A lighter exposure of the abundant signal in the let-7(mn112) lane shows a faster mobility for both bands compared to the position of the bands detected in RNA from wild type, as expected to account for the 193-nt deletion in the let-7(mn112) gene.

(C) The same RNA samples as in A were subjected to RT-PCR using oligo dT in the RT reaction and the let-7-specific primers A63 + A62 (first panel), A127 + A62 (second panel), or forward primer consisting of the SL1 sequence plus A62 (third panel) for PCR.

Figure 3. Predicted structures of primary and SL1-spliced let-7 transcripts.

(A) Potential secondary structures of the 890-nt primary let-7 transcript and

(B) the SL1-spliced RNA were generated using the mfold program (Zuker 2003). The structures shown are the lowest free-energy predictions. The SL1 splice site, SL1 donor sequence, and mature let-7 RNA are outlined and labeled. The boxes indicate predicted structural rearrangements surrounding the let-7 stem loop region resulting from SL1 splicing.

Temporally regulated transcription and processing of let-7 primary transcripts

Accumulation and trans-splicing of the predicted let-7 primary transcripts precede the appearance of mature let-7 RNA. In developmentally staged RNA samples, the 22-nt let-7 RNA first appears late in the third larval stage (L3), accumulates in L4, and persists to adulthood (Fig. 4A; Reinhart et al. 2000; Johnson et al. 2003). Similarly, the let-7 primary transcripts and SL1-spliced form were virtually undetectable by RT-PCR analyses until the L3 stage of development (Fig. 4B). The relatively higher levels of these transcripts at L3 compared to later stages indicates that there may be a burst of transcriptional activity in L3 and subsequent processing that produces mature let-7 RNA that is maintained through the last larval and adult stages.

Temporally regulated expression of let-7 RNAs

Figure 4. Temporally regulated expression of let-7 RNAs.

(A) Total RNA from developmentally staged wild-type worms was subjected to Northern analyses to detect the precursor and mature let-7 RNAs as described in Figure 2A.

(B) The same RNA samples as in A were subjected to RT-PCR to detect let-7 primary transcript and control eft-2 mRNA (upper panel) or the SL1-appended let-7 RNA (lower panel).

The ribonuclease III-type enzymes Drosha and Dicer and members of the Argonaute family function to produce mature miRNAs (Grishok et al. 2001; Hutvagner et al. 2001; Ketting et al. 2001; Hutvagner and Zamore 2002; Mourelatos et al. 2002; Lee et al. 2003). We tested whether depletion of these factors would affect the expression of the primary let-7 transcripts that enter the processing pathway. Drosha is a nuclear RNase required for efficient production of precursor miRNAs from primary transcripts in human cells (Lee et al. 2003). The C. elegans gene F26E4.10 is a predicted homolog of Drosha (Lee et al. 2003). If F26E4.10 processes miRNA primary transcripts, then let-7 pri-miRNAs might accumulate upon depletion of this putative RNase. In contrast, the RNase III Dicer, encoded by dcr-1 in C. elegans, functions downstream from Drosha processing and, thus, loss of its activity should not affect processing of let-7 primary transcripts. The rrf-3(pk1426)C. elegans strain, which is hypersensitive to RNA interference (RNAi; Simmer et al. 2002), was subjected to RNAi targeting F26E4.10 or dcr-1. This procedure resulted in the specific reduction of dcr-1 or F26E4.10 mRNA levels (Fig. 5A). RNAi depletion of F26E4.10 or dcr-1 apparently had no effect on expression of unspliced let-7primary transcripts (Fig. 5A, pri-let-7 panel). As expected, worms undergoing RNAi against dcr-1 accumulated let-7 precursor RNA (Fig. 5A, pre-let-7 panel). Interestingly, an increase in the SL1-let-7 product was specifically observed in worms depleted of F26E4.10 (Fig. 5A, SL1-let-7 panel), indicating that the trans-spliced form of let-7 is a substrate for processing by the putative Drosha homolog.

Depletion of Drosha, Dicer, or Argonaute homologs inhibits let-7 maturation at independent steps

Figure 5. Depletion of Drosha, Dicer, or Argonaute homologs inhibits let-7 maturation at independent steps.

(A) Total RNA from worms fed vector control (lane 1), F26E4.10 (Drosha) dsRNA (lane 2), or dcr-1 (Dicer) dsRNA (lane 3) expressing bacteria was isolated, subjected to Northern analyses to detect precursor and mature let-7 or to oligo dT reverse transcription, followed by PCR amplification of let-7 transcripts, and mRNAs for F26E4.10, dcr-1, and eft-2, as indicated. 5S rRNA detected by ethidium bromide staining serves as a control for the Northern analyses.

(B) Total RNA from worms fed vector control (lane 1) or alg-1 (argonaute like gene 1) dsRNA (lane 2) expressing bacteria was isolated, subjected to Northern analyses to detect precursor and mature let-7 or to oligo dT reverse transcription, followed by PCR amplification of let-7 transcripts, and mRNAs for alg-1 and eft-2, as indicated. 5S rRNA detected by ethidium bromide staining serves as a control for the Northern analyses.

Transcription of let-7 primary RNAs

This study reports one of the first complete primary transcripts for an animal microRNA gene. The proposal that the ~1731-nt and ~890-nt RNAs serve as let-7 primary transcripts is supported by the observation that these RNAs stably accumulate in let-7 deletion mutants that produce compromised levels of mature let-7. Additionally, the timing of expression of the primary transcripts correlates well with the temporal appearance of 22-nt let-7 RNA in vivo.

The let-7 primary transcripts in C. elegans have the hallmarks of Pol-II-directed synthesis—they are apparently 5'-end capped, 3'-end polyadenylated, and substrates for splicing. However, it is yet to be experimentally determined which polymerase is responsible for transcribing let-7 or any miRNA gene. A recent report from the Slack laboratory defined a regulatory element responsible for restricting let-7 expression in the hypodermis to the last larval and adult stages (Johnson et al. 2003). A 116-bp region located ~1200 nt upstream of the mature let-7 RNA coding sequence was found to be essential for full let-7 activity. Interestingly, this region, called the temporal regulatory element (TRE), can also restrict hypodermal expression of a protein-coding gene to the same time in development as the appearance of let-7 RNA (Johnson et al. 2003). The TRE is ~200 nt upstream of the initiation site for transcription of the longest let-7 primary transcript. It remains to be determined whether the TRE regulates the expression of either or both of the endogenous pri-let-7 transcripts. In addition to the TRE, the let-7 genes in C. elegans and C. briggsae also share a highly homologous region ~200 nt upstream of the transcriptional start site for the ~890-nt pri-let-7 RNA. Perhaps independent regulatory elements afford tissue as well as temporally regulated transcription of let-7 RNAs.

Our demonstration that let-7 primary transcripts in C. elegans are polyadenylated and undergo splicing provides additional support to the indirect evidence that at least some miRNAs are transcribed by Pol II (Bartel 2004): Predicted miRNA promoter regions can produce temporally or spatially regulated expression of proteins (Johnson et al. 2003; Johnston and Hobert 2003), some miRNA sequences match ESTs (Lagos-Quintana et al. 2002; Aukerman and Sakai 2003; Smalheiser 2003), long miRNA primary transcripts might not be supported by Pol III, and some miRNAs can be processed from heterologous Pol II synthesized transcripts (Zeng et al. 2002; Zeng and Cullen 2003). If Pol II generally directs expression of miRNA genes, then how are pri-miRNA transcripts distinguished from messenger RNAs (mRNAs) in the cell? Usually, capping, splicing, and polyadenylation events facilitate export of mRNAs to the cytoplasm for translation to protein (Reed 2003). Pri-miRNAs may contain sequence or structural elements that direct them to the nuclear processing pathways instead of the mRNA export route. For some miRNAs, efficient excision of the precursor hairpin may be sufficient to circumvent export of the pri-miRNA substrate.

Stepwise maturation of let-7 miRNA

The elaborate transcription and processing steps expended to eventually yield a 22-nt RNA could provide additional levels for regulation. Alteration of pri-miRNA sequence or structure beyond the precursor hairpin can affect the processing or stability of the RNA in vitro or in vivo when expressed ectopically from a transgene (Lee et al. 2003;Zeng and Cullen 2003; Chen et al. 2004). Additionally, depletion of Drosha, Dicer, Argonaute, and Exportin-5 proteins can interfere with specific steps of miRNA biogenesis (Grishok et al. 2001; Hutvagner et al. 2001; Ketting et al. 2001; Lee et al. 2003; Yi et al. 2003; Bohnsack et al. 2004; Lund et al. 2004). We show that the let-7 primary transcripts, SL1-appended form, and precursor RNA can each stably accumulate in vivo when distinct processing steps are blocked by alteration of the RNA sequence or by depletion of transacting factors (Figs. 1, 2, 5), contributing to the proposal that miRNA expression can be regulated at the level of processing (Lee et al. 2002, 2003). For example, the mature form of C. elegans miR-38 is primarily observed in embryos but its precursor is detectable at all stages of development (Ambros et al. 2003).

The specific accumulation of the SL1-spliced form of let-7 upon depletion of the predicted C. elegans Drosha homolog, F26E4.10 (Fig. 5A), indicates that this form of let-7 is a substrate for the Drosha processing pathway in C. elegans. Inactivation of F26E4.10 by feeding RNAi produces very mild phenotypes, and we detect little if any decrease in the accumulation of mature let-7 RNA compared to that detected in controls (Fig. 5A, mature let-7 panel). It is possible that there is a limited capacity for the amount of mature let-7 RNA maintained in the cell, and that only a fraction of the primary and precursor substrates generate stable mature RNA. This idea is supported by the observation that relative levels of precursor miRNA in dcr-1-depleted animals can greatly exceed the amount of mature miRNA detected in wild-type animals (Fig. 5A). Additionally, injection of dsRNA targeting F26E4.10 also failed to generate obvious developmental phenotypes that would imply inadequate miRNA processing (data not shown). Although the mRNA levels of F26E4.10 were effectively depleted by RNAi (Fig. 5A), the remaining protein activity is unknown. Thus, it is presently unclear if adequate F26E4.10 activity remains under the RNAi conditions or if other factors compensate in the miRNA biogenesis pathway.

Depletion of alg-1 by RNAi results in a significant reduction in the accumulation of let-7 and lin-4 mature RNAs (Grishok et al. 2001). In the case of lin-4, this defect can be attributed to inefficient processing of pre-lin-4 (Grishok et al. 2001). However, there is no apparent precursor buildup for let-7 (Fig. 5B; Grishok et al. 2001). Because dcr-1 (RNAi) causes accumulation of the precursors for both miRNAs (Grishok et al. 2001), there is no evidence that pre-let-7 is particularly unstable in vivo. Our data indicate that transcription and trans-splicing of the initial let-7 RNAs are unaffected in alg-1 (RNAi) (Fig. 5B), ruling out the possibility that reduction of mature let-7 results from significant defects in these processes. Because a variety of mature miRNAs have been found in complexes containing argonaute homologs (Hammond et al. 2001; Hutvagner and Zamore 2002; Mourelatos et al. 2002), these proteins may function primarily to regulate the single-stranded 22-nt forms of miRNAs.

Splicing of let-7 primary transcripts

Trans-splicing of Spliced Leader (SL) sequences to the 5' end of mRNAs is a common phenomenon in C. elegans (Blumenthal 1995). Despite the observation that about 70% of C. elegans pre-mRNAs undergo this process, a definitive function for trans-splicing is yet to be revealed. Nonetheless, deletion of genes encoding the SL1 RNA produces embryonic lethality (Ferguson et al. 1996), and it has been proposed that SL1 trans-splicing may help optimize translation of some mRNAs (Blumenthal 1995). If trans-splicing by SL1 RNA onto some pri-miRNAs influences processing, then miRNA biogenesis could be another important function of trans-splicing in C. elegans.

Conservation of the trans-splice site in C. elegans and C. briggsae and the demonstration that removal of the splice site compromises the rescuing activity of the let-7 gene suggest that trans-splicing may be an important step in the biogenesis of let-7 RNA in C. elegans. What function could trans-splicing serve in the let-7 processing pathway? Sequence or structural alteration of let-7 primary transcripts may be important for subsequent processing steps. The predicted secondary structures produced by folding the entire 890-nt let-7 RNA, or the longer 1731-nt RNA (data not shown), and SL1 spliced version reveal possible structural alterations immediately adjacent to the stem-loop precursor containing the mature RNA (Fig. 3, boxes). Addition of the SL1 22 nt to the 5' end of the let-7RNA supports the formation of an extended stem that is initiated by complementary base-pairing between the SL1 sequences and nucleotides encoded by let-7. These predicted structural rearrangements are conserved in the C. briggsaelet-7 gene. Additionally, splicing of the let-7 primary transcripts removes sequences that could be inhibitory to the miRNA processing machinery.

Sequences and structures flanking the miRNA hairpin influence maturation to the 22-nt form (Lee et al. 2003; Zeng and Cullen 2003; Chen et al. 2004). Splicing of miRNA primary transcripts could significantly change the nucleotide and structural elements surrounding the hairpin precursor. Although ectopic expression of truncated miRNA transcripts supports production of the mature form of some miRNAs (Zeng and Cullen 2003; Chen et al. 2004), the natural pri-miRNA transcripts may include sequences or structures that regulate expression of specific miRNAs. Thus, identification of endogenous pri-miRNA transcripts is important for understanding how members of this new RNA family are recognized in vivo as substrates for the miRNA processing pathway.

Materials and Methods:

RT-PCR analyses
Total RNA from indicated stages of worm strains was extracted using TRIzol reagent (GIBCO-BRL), including a deoxyribonuclease step (RQ1 DNase, Promega). First-strand cDNA synthesis was performed with 5 µg RNA and SUPERSCRIPT II (Invitrogen) reagents, according to the manufacturer’s recommendations. RNA ligase-mediated rapid amplification of 5' and 3' cDNA ends (RLM-RACE) was accomplished with the GeneRacer Kit (Invitrogen), using 5 µg of total RNA from L3 stage worms. Primer sequences of oligonucleotides used in the RT-PCR reactions are as follows:

A62: 5'-GGCTCCATGGATACATTACTCAACAG-3'

A63: 5'-GGATCATCAATCAAGTGTGCACTG-3'

A76: 5'-TGTGAGAGAGCAAGACGACGCAGCTTCG-3'

A127: 5'-GAGTAGCCCACCTAGCAGCGGTCG-3'

A90 (SL1): 5'-GGTTTAATTACCCAAGTTTGAG-3'

A112 (F-F26E4.10): 5'-TCAGATGTTCCAGCTCGTTCTTCG-3'

A113 (R-F26E4.10): 5'-CAGCATTGCCATTTGGAGATTCTCC-3'

A211 (F-dcr-1): 5'-GCTGCAGATGGAATGTCATTAGAGAGATTC-3'

A212 (R-dcr-1): 5'-AGTAGCTTTCGCGATTCGATAGTTCCTTCC-3'

A116 (F-eft-2): 5'-GGTCAACTTCACGGTCGATG-3'

A117 (R-eft-2): 5'-TCCGAGCTTCTCAACAAGAGC-3'

A110 (F-alg-1): 5'-CAAGTGGACCGATTAGTTCGACG-3'

A111 (R-alg-1): 5'-TCATTGCTGGGTTGATGGTGATT-3'.

Northern analyses
Detection of small RNA species (<200 nt) was carried out by polyacrylamide Northern methods, as previously detailed (Pasquinelli et al. 2003). Analyses of larger RNAs were performed by separating 8 µg of total RNA in 1.2% agarose gels under denaturing conditions. RNA was transferred and UV cross-linked to nylon membranes (Zeta-Probe GT, Biorad), which were then hybridized to PCR-generated DNA probe (A63 + A62) radiolabeled using Prime-It II Random Primer Labeling Kit (Stratagene). Hybridization and wash conditions were performed as previously described (Pasquinelli et al. 2003) except that procedures were carried out at 58°C.

Transgenics
For rescue experiments, the wild-type (pAEP19) or 3' splice site deleted (pSEH6) clones were injected at 5 ng/µL with a GFP marker construct (myo-2::yC2.1) into the SP231 (mnDp1(X/V)/+ V; unc-3(e151)let-7(mn112) X) strain (Meneely and Herman 1979). Transgenic, coiling, homozygous let-7(mn112) progeny were scored for rescue: The wild-type construct rescued the lethality in 100/101 of the transgenics; the pSEH6 construct showed no (0/94) rescuing activity. Expression of pri-let-7 from pSEH6 was verified by performing RT-PCR from RNA isolated from trans-genic worms; the RNA encoded by the transgene is 187 nt longer than that synthesized by the let-7(mn112) allele and is thus distinguishable from the genomic expression of let-7(mn112).

RNAi
The rrf-3(pk1426) L4 stage worms were cultured on RNAi plates seeded with bacteria containing vector control plasmid or plasmids expressing double-stranded RNA corresponding to the F26E4.10 or dcr-1 or alg-1 genes (Kamath et al. 2003). Progeny from these worms were reared on the same type of RNAi food and collected at the L4 stage of development for RNA isolation and analyses.

Acknowledgments:

We thank members of our laboratory and J. Posakony, W. Mc-Ginnis, J. Dahlberg, and E. Lund for suggestions and critical reading of the manuscript. J.B. and S.H. were supported by funds from the National Institutes of Health (NIH) Cellular and Molecular Biology Graduate Student Training Grant.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

Footnotes:

¹ These authors contributed equally.

Article published online ahead of print. Article and publication date are at
http://www.rnajournal.org/cgi/doi/10.1261/rna.7122604.

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This detailed study of let-7 microRNAs by John Bracht, Shaun Hunter, Rachel Eachus, Phillip Weeks, and Amy  Pasquinelli reveals for the first time that at the point of transcription, the primary microRNA is a quite long RNA molecule of 890 nt -1731 nt length, and that the 5' leader intron is spliced co-incident with transcription, and may itself be further used in gene regulation.

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