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Trans-Splicing of the C. Elegans Let-7 Primary Transcript Developmentally © 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev182212. doi:10.1242/dev.182212 CORRECTION Correction: Trans-splicing of the C. elegans let-7 primary transcript developmentally regulates let-7 microRNA biogenesis and let-7 family microRNA activity (doi: 10.1242/dev.172031) Charles Nelson and Victor Ambros There was an error published in Development (2019) 146, dev172031 (doi:10.1242/dev.172031). In Table S3 in the supplementary material, the genotype for strain VT3903 was incorrect. Corrected: maEx267 [rol-6(su1006); unc-119+, plet-7::let-7 locus without mature let-7 sequence]; let-7(ma388) X; mir-48(n4097), maIs105 V Original: maEx264 [unc-119+; plet-7::let-7 locus without mature let-7]; let-7(ma388) X; mir-48(n4097), maIs105 V The online supplementary PDF has been been updated. The authors apologise for this error and any inconvenience it may have caused. DEVELOPMENT 1 © 2019. Published by The Company of Biologists Ltd | Development (2019) 146, dev172031. doi:10.1242/dev.172031 RESEARCH ARTICLE Trans-splicing of the C. elegans let-7 primary transcript developmentally regulates let-7 microRNA biogenesis and let-7 family microRNA activity Charles Nelson and Victor Ambros* ABSTRACT promotes the differentiation of cellular fates (Reinhart et al., 2000). The sequence and roles in developmental progression of the Orthologs of the C. elegans let-7 microRNA are easily identified microRNA let-7 are conserved. In general, transcription of the let-7 across animal phyla because of the near perfect conservation of the primary transcript ( pri-let-7) occurs early in development, whereas entire 22 nt sequence (Pasquinelli et al., 2000). In many species, processing of the mature let-7 microRNA arises during cellular let-7 paralogs encode additional family members, including mir-48, differentiation. In Caenorhabditis elegans and other animals, the mir-84 and mir-241 in C. elegans, which differ from let-7 in some RNA-binding protein LIN-28 post-transcriptionally inhibits let-7 of their non-seed nucleotides. In C. elegans, let-7 seed family biogenesis at early developmental stages, but the mechanisms by (let-7fam) microRNAs function semi-redundantly to regulate stage- which LIN-28 does this are not fully understood. Nor is it understood specific larval cell fate transitions, with mir-48, mir-84 and mir-241 how the developmental regulation of let-7 might influence the primarily promoting the L2-to-L3 transition and let-7 primarily expression or activities of other microRNAs of the same seed promoting the L4-to-adult transition (Reinhart et al., 2000; Abbott family. Here, we show that pri-let-7 is trans-spliced to the SL1 splice et al., 2005). leader downstream of the let-7 precursor stem-loop, which produces In C. elegans, two major primary transcripts of let-7 ( pri-let-7) a short polyadenylated downstream mRNA, and that this trans- are produced, pri-let-7 A and pri-let-7 B of 1731 and 890 ′ splicing event negatively impacts the biogenesis of mature let-7 nucleotides, respectively. The 5 end of these transcripts can be microRNA in cis. Moreover, this trans-spliced mRNA contains further processed by trans-splicing with a 22-nucleotide splice sequences that are complementary to multiple members of the let-7 leader (SL) RNA (SL1) to produce the 728 nucleotide SL1-pri-let-7 seed family (let-7fam) and negatively regulates let-7fam function in (Bracht et al., 2004; Van Wynsberghe et al., 2011), (Fig. 1A). All trans. Thus, this study provides evidence for a mechanism by which three of these pri-let-7 transcripts contain the let-7 precursor hairpin splicing of a microRNA primary transcript can negatively regulate said plus additional downstream sequences, which include an element microRNA in cis as well as other microRNAs in trans. with complementarity to the let-7fam seed sequence that has been shown to associate in vivo with the Argonaute protein, ALG-1 KEY WORDS: let-7 family microRNAs, lin-28, Trans-splicing, (Zisoulis et al., 2012). Developmental timing, Caenorhabditis elegans pri-let-7 transcripts are expressed at all four larval stages of C. elegans development (L1-L4), whereas mature let-7 is abundantly INTRODUCTION expressed only in the L3 and L4 larval stages. Intriguingly, pri-let-7 MicroRNAs are endogenous ∼22 nt RNAs that are enzymatically levels oscillate during each larval stage, peaking mid-stage and processed from longer primary transcripts, and that repress protein dipping during larval molts, likely as a result of underlying pulsatile expression through imperfect base pairing with their target mRNAs. transcriptional activity of the let-7 locus (Van Wynsberghe et al., Nucleotides 2-8 of the microRNA, known as the seed, instigate 2011; McCulloch and Rougvie, 2014; Perales et al., 2014; Van target recognition through essentially complete complementarity, Wynsberghe et al., 2014). Why pri-let-7 pulses with each larval stage whereas base pairing via the non-seed nucleotides (9-22 of the remains unclear. However, the distinct developmental profiles of microRNA) is less constrained than is seed pairing (He and Hannon, pri-let-7 and mature let-7, particularly at early larval stages, indicate 2004). microRNAs that contain an identical seed sequence but potent post-transcriptional inhibition of let-7 biogenesis during the differ in their non-seed nucleotides are classified together as a L1 and L2 stages. ‘family’ based on their presumed evolutionary relatedness, and their LIN-28 is a conserved RNA-binding protein that can bind to and potential to act in combination on the same targets (Bartel, 2009; regulate a variety of RNAs (Stefani et al., 2015; Wilbert et al., 2012). Ambros and Ruvkun, 2018). In C. elegans, LIN-28 is expressed at early larval stages and exerts a The let-7 gene was initially identified in a screen for strong inhibition of let-7 processing (Van Wynsberghe et al., 2011). developmental defects in Caenorhabditis elegans (Meneely and Similar to C. elegans, mammalian Lin28 inhibits let-7 processing Herman, 1979), and later found to encode a microRNA that and is expressed in more pluripotent cells. Extensive studies in mammalian systems have also shown that Lin28 exerts its inhibition of let-7 by binding to the stem-loop of either pri-let-7 or pre-let-7 to Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. directly inhibit processing (reviewed by Tsialikas, 2015). Although CLIPseq data from C. elegans indicated that, similar to in mammals, *Author for correspondence ([email protected]) LIN-28 could bind pri-let-7 in vivo to inhibit accumulation of mature V.A., 0000-0002-5562-5345 let-7 microRNA, binding occurs downstream of the let-7 stem-loop, which suggests a different mode of regulation on let-7, the precise Received 17 September 2018; Accepted 11 February 2019 mechanism(s) of which remains unclear (Stefani et al., 2015). DEVELOPMENT 1 RESEARCH ARTICLE Development (2019) 146, dev172031. doi:10.1242/dev.172031 Fig. 1. The C. elegans let-7 locus produces four transcripts. (A) Major transcripts produced from the C. elegans let-7 locus, and the locations of probes used in the northern blots shown in C, and in subsequent figures. (B) Non-qRT-PCR analysis of total RNA from a mix-population of wild-type (WT) animals, using an SL1 forward primer and LCE reverse primer, with (+) and without (−) reverse transcriptase (RT) in the cDNA synthesis reaction. Numbers mark dsDNA ladder bands in bp. (C) Total RNA from WT and LCE-deletion mid-L2 animals (20 h after plating) analyzed using northern blotting with probes for the LCE and SL1-LCE. Numbers mark known RNA sizes in nt: 872 nt is in vitro transcribed pri-let-7 B, 244 nt is in vitro transcribed SL1-LCE and 153 nt is 5.8s rRNA. Numbers with a tilde before them were estimated from known RNA sizes. Trans-splicing is the act of joining two separate RNAs. In we have characterized a splicing event that involves let-7 primary C. elegans, ∼70% of all transcripts (including mRNAs and transcripts and that can regulate let-7 biogenesis in cis as well as microRNA primary transcripts) are trans-spliced with an SL let-7fam activity in trans. RNA. The outcomes of trans-splicing include separating the individual mRNAs of polycistronic operon transcripts, shortening RESULTS the 5′ untranslated regions (5′ UTRs) of RNAs and changing the 5′ The let-7 locus produces a short trans-spliced transcript that RNA cap from monomethyl to trimethyl guanosine. There are two contains LCSs major classes of SL RNAs with distinct sequences: SL1 and SL2. As mentioned above, a previous study identified a region of pri-let-7 Although there are exceptions, SL1 trans-splicing tends to be the in C. elegans that contains a let-7 complementary element (LCE) most common 5′ trans-splicing event of a primary RNA transcript (Zisoulis et al., 2012). We confirmed that three sites within the LCE and invariably results in rapid degradation of the 5′ ‘outron’. SL2 element have complementarity to let-7fam microRNAs so as to trans-splicing is generally restricted to downstream open reading permit base pairing to the let-7-family seed sequence plus varying frames (ORFs) of operons after SL1 trans-splicing and degrees of 3′ supplemental pairing (Fig. S1A). We also noted that an polyadenylation of the first mRNA (Morton and Blumenthal, additional transcript, C05G5.7, is annotated to be transcribed from 2011; Blumenthal, 2012). Similar to cis-splicing, trans-splicing in the let-7 locus and to contain a 647 nt 5′ UTR that includes the let-7 C. elegans uses a consensus acceptor sequence of ‘TTTCAG’ stem-loop, a 111 nt ORF that contains the LCE, and a 79 bp 3′ UTR.
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