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A stem–loop and a UAYYUU are necessary for SL2 trans -splicing. ( A , top ) Diagram of Ur element stem mutation in vitro splicing substrates. D st/lp is a deletion of the 17 nt in the stem–loop. The sequence of each stem–loop substitution is shown next to the wild-type stem–loop, with shaded boxes indicating changed nucleotides. ( Bottom ) RT–PCR of in vitro splicing reactions. ( B , top ) Diagram of Ur element UAYYUU mutation in vitro splicing substrates. Horizontal bars below the sequence indicate the nucleotides of the Ur element UAYYUU and the four overlapping downstream mismatched copies. G1 and G2 contain substitutions in the first full UAYYUU motif, G3 contains substitutions in the four downstream mismatched UAYYUU copies, and G1/G3 and G2/G3 contain substitutions in all of them. ( Bottom ) RT–PCR of in vitro splicing reactions. ( C ) Bioinformatic analysis comparing the percentages of genes within (column A) or not within (column B) operons that contain multiple copies of the sequence motif UAYYUU (allowing a 1-nt mismatch), within a 20-nt window located 40–60 nt downstream from 3 9 end cleavage sites (cs). Motif copy # indicates the number of instances of the motif occurring within the window. Motif copies may overlap.
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Spliced leader (SL) trans-splicing in Caenorhabditis elegans attaches a 22-nucleotide (nt) exon onto the 5' end of many mRNAs. A particular class of SL, SL2, splices mRNAs of downstream operon genes. Here we use an embryonic extract-based in vitro splicing system to show that SL2 specificity information is encoded within the polycistronic pre-mRNA,...
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... order to examine the roles of the two parts of the Ur element, we created additional targeted substitution and deletion mutation constructs. As shown in Figure 5A, a precise deletion of the predicted stem results in loss of SL2 trans-splicing (19% of wild type). This may indicate that the stem is necessary for some aspect of trans- splicing, or that it is important for the integrity of the downstream RNA following upstream gene 39 end cleavage. ...
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... (19% of wild type). This may indicate that the stem is necessary for some aspect of trans- splicing, or that it is important for the integrity of the downstream RNA following upstream gene 39 end cleavage. We also replaced the wild-type stem-loop with different heterologous stem-loops, including one that is predicted to be quite stable (Fig. 5A, st/lp 1) and three that maintain the wild-type loop sequence, altering only 3 or 4 bp within the stem. Substitution with any of these stem-loops increases SL2 trans-splicing compared with the deletion, but wild-type levels are never com- pletely restored. This is consistent with the idea that a stem-loop is required (for ...
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... rla-1 ICR contains multiple, overlapping copies of UAYYUU, some with a single nucleotide mismatch (underlined in Fig. 5B, top). In fact, the majority of Ur ele- ments in other operons have multiple copies of UAYYUU in this small region ( Fig. 5C; data not shown). To assess the contribution of these ''extra'' copies, we created constructs that contained substitutions within the first full UAYYUU (Fig. 5B, G1 and G2), within the four downstream mis- matched ...
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... rla-1 ICR contains multiple, overlapping copies of UAYYUU, some with a single nucleotide mismatch (underlined in Fig. 5B, top). In fact, the majority of Ur ele- ments in other operons have multiple copies of UAYYUU in this small region ( Fig. 5C; data not shown). To assess the contribution of these ''extra'' copies, we created constructs that contained substitutions within the first full UAYYUU (Fig. 5B, G1 and G2), within the four downstream mis- matched UAYYUU copies (Fig. 5B, G3), or within all the copies combined. G1 and G2 demonstrate that 2-nt sub- stitutions within the most 59 UAYYUU ...
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... In fact, the majority of Ur ele- ments in other operons have multiple copies of UAYYUU in this small region ( Fig. 5C; data not shown). To assess the contribution of these ''extra'' copies, we created constructs that contained substitutions within the first full UAYYUU (Fig. 5B, G1 and G2), within the four downstream mis- matched UAYYUU copies (Fig. 5B, G3), or within all the copies combined. G1 and G2 demonstrate that 2-nt sub- stitutions within the most 59 UAYYUU reduce, but do not eliminate, SL2 trans-splicing. The same is true where all downstream copies are mutated (Fig. 5B, G3). Importantly, when the mutations are combined (Fig. 5B, G1/G3 or G2/G3), SL2 trans-splicing is almost ...
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... within the first full UAYYUU (Fig. 5B, G1 and G2), within the four downstream mis- matched UAYYUU copies (Fig. 5B, G3), or within all the copies combined. G1 and G2 demonstrate that 2-nt sub- stitutions within the most 59 UAYYUU reduce, but do not eliminate, SL2 trans-splicing. The same is true where all downstream copies are mutated (Fig. 5B, G3). Importantly, when the mutations are combined (Fig. 5B, G1/G3 or G2/G3), SL2 trans-splicing is almost completely lost (14% and 12% wild type). This demonstrates that the additional mismatched copies act additively in SL2 trans- splicing, and are partially ...
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... ask whether partial redundancy was widespread among ICRs, we searched for the UAYYUU motif or any single nucleotide mismatch within a specified region (40- 60 nt downstream from 39 end cleavage sites). Figure 5C shows that 75% of operon ICRs (column A) have at least one UAYYUU (allowing a single mismatch) within that limited region, while only 37% of nonoperon genes do (column B). Furthermore, operons are far more likely to have multiple copies. ...
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... unaffected. Instead, we got unex- pected, but quite interesting, results. The long Ur ele- ment RNA oligonucleotide is depicted in Figure 6A. It is nt long and contains the stem and all copies of the UAYYUU sequence. An oligonucleotide containing sub- stitutions within the predicted stem and each of the UAYYUU copies (the G1/G3 substitutions in Fig. 5B) was used as a control. In each splicing reaction, the RNA oligonucleotide was added immediately prior to addition of the RNA substrate. We first tested a range of oligonu- cleotide concentrations (0-333 ng/mL), and determined that nonspecific total splicing inhibition occurred at the highest concentrations (data not shown). However, ...
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... among species still main- tain stem-forming potential. Third, the in vivo mutagen- esis of gpd-3 and the in vitro experiments of rla-1 demonstrate that different stem-loop sequences recov- ered partial SL2 trans-splicing compared with the con- struct in which the stem-loop was deleted and substitu- tions were not predicted to form stems ( Fig. 5A; data not shown). Lack of full recovery by heterologous stem-loops may indicate that subtle structural or sequence aspects of the region are playing a role. Perhaps a stem-loop supplies a binding surface, but some feature of the sequence provides the ideal context. We propose that one purpose of the stem is to add a discriminating ...
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... It is a critical element in the generation of mRNAs derived from genes situated downstream from the first gene in an operon, since the spliced leader RNA provides the 5 ′ cap for such mRNAs, allowing the transspliced mRNAs to be recognized by the translation machinery. Addition of the spliced leader is also thought to prevent termination of transcription following polyadenylation of the upstream mRNA (Evans et al. 2001;Haenni et al. 2009;Lasda et al. 2010). Thus, spliced leader transsplicing significantly facilitates the evolution of eukaryotic operons. ...
... Previous studies in C. elegans have identified the presence of conserved elements, termed Ur motifs, that are im-plicated in SL2 trans-splicing and are expected to be present about 50 bp upstream of the trans-splice site (Lasda et al. 2010). Such elements have also been detected in putative T. spiralis operons (Pettitt et al. 2014). ...
... However, we cannot exclude the possibility that the sequence conservation we observe is due to convergent evolution, with SL2-type trans-splicing having independently arisen in the lineages leading to Clade V and Clade I nematodes. We think that this second scenario is less likely, as the convergent evolution of the stem-loop 3 motif would need to be accompanied by parallel coevolution of the interaction with the polyadenylation machinery (Evans and Blumenthal 2000;Evans et al. 2001;Lasda et al. 2010). Expanding our analysis into the other nematode clades could address this uncertainty. ...
Spliced leader trans-splicing is essential for the processing and translation of polycistronic RNAs generated by eukaryotic operons. In C. elegans, a specialised spliced leader, SL2, provides the 5' end for uncapped pre-mRNAs derived from polycistronic RNAs. Studies of other nematodes suggested that SL2-type trans-splicing is a relatively recent innovation, confined to Rhabditina, the clade containing C. elegans and its close relatives. Here we conduct a survey of transcriptome-wide spliced leader trans-splicing in Trichinella spiralis, a distant relative of C. elegans with a particularly diverse repertoire of 15 spliced leaders. By systematically comparing the genomic context of trans-splicing events for each spliced leader, we identified a subset of T. spiralis spliced leaders that are specifically used to process polycistronic RNAs - the first examples of SL2-type spliced leaders outside of Rhabditina. These T. spiralis spliced leader RNAs possess a perfectly conserved stem-loop motif previously shown to be essential for SL2-type trans-splicing in C. elegans We show that genes trans-spliced to these SL2-type spliced leaders are organised in operonic fashion, with short intercistronic distances and a degree of homology with C.elegans operons. Our work substantially revises our understanding of nematode spliced leader trans-splicing, showing that SL2 trans-splicing is a major mechanism for nematode polycistronic RNA processing, which may have evolved prior to the radiation of the nematode phylum. This work has important implications for the improvement of genome annotation pipelines in nematodes and other eukaryotes with operonic gene organisation.
... Briefly, cleavage and polyadenylation of the upstream mRNA occurs by a conventional mechanism (Garrido-Lecca et al. 2016), and the resulting 59-phosphate end of the nascent RNA is subjected to XRN-2-mediated degradation (Miki et al. 2016). The downstream pre-mRNA is, however, protected from XRN-2 by a U-rich (Ur) element proposed to recruit SL2 snRNP for trans-splicing (Graber et al. 2007;Lasda et al. 2010). Recently, it has been shown that expression of xrn-2, a downstream gene of the rpl-43xrn-2 operon, is negatively auto-regulated by XRN-2 activity in vivo (Miki et al. 2016); XRN-2 may compete with transcribing Pol II and/or the trans-splicing machinery to terminate transcription by the "torpedo" mechanism without affecting expression of the upstream rpl-43 gene (Miki et al. 2016). ...
While DNA serves as the blueprint of life, the distinct functions of each cell are determined by the dynamic expression of genes from the static genome. The amount and specific sequences of RNAs expressed in a given cell involves a number of regulated processes including RNA synthesis (transcription), processing, splicing, modification, polyadenylation, stability, translation, and degradation. As errors during mRNA production can create gene products that are deleterious to the organism, quality control mechanisms exist to survey and remove errors in mRNA expression and processing. Here, we will provide an overview of mRNA processing and quality control mechanisms that occur in Caenorhabditis elegans , with a focus on those that occur on protein-coding genes after transcription initiation. In addition, we will describe the genetic and technical approaches that have allowed studies in C. elegans to reveal important mechanistic insight into these processes.
... Spliced leader trans-splicing is an essential element in the generation of mRNAs derived from genes situated downstream of the first gene in an operon since the spliced leader RNA provides the 5' cap for such mRNAs, allowing the trans-spliced mRNAs to be recognised by the translation machinery. Addition of the spliced leader is also thought to prevent termination of transcription following polyadenylation of the upstream mRNA (Evans et al. 2001;Lasda et al. 2010). Thus, spliced leader trans-splicing is an essential prerequisite for the evolution of eukaryotic operons. ...
... doi: bioRxiv preprint first posted online May. 17, 2019; 1 downstream pre-mRNA (Liu et al. 2003;Huang et al. 2001;Lasda et al. 2010). Moreover, previous studies have shown that at least one of these T. spiralis intercistronic regions is recognised by C. elegans SL2 trans-splicing (Pettitt et al. 2014), further supporting the functional conservation of the process between the two nematodes. ...
... However, credible operons have been identified in a member of this clade, Brugia malayi (Ghedin et al. 2007;Guiliano and Blaxter 2006), and putative intercistronic regions have been characterised (Liu et al. 2010), including a potential Ur element, which was shown to be important for the expression of the downstream gene in a transgenic assay (Liu et al. 2010). This work showed that the Ur element behaves similarly to Ur elements in C. elegans polycistronic RNAs, and since Ur elements are specifically involved in recruiting SL2 RNAs to downstream pre-mRNAs (Liu et al. 2003;Lasda et al. 2010), this suggests that intercistronic region-specific spliced leader RNAs also exist in B. ...
Spliced leader trans -splicing is intimately associated with the presence of eukaryotic operons, allowing the processing of polycistronic RNAs into individual mRNAs. Most of our understanding of spliced leader trans -splicing as it relates to operon gene expression comes from studies in C. elegans . In this organism, two distinct spliced leader trans -splicing events are recognised: SL1, which is used to replace the 5′ ends of pre-mRNAs that have a nascent monomethyl guanosine cap; and SL2, which provides the 5′ end to uncapped pre-mRNAs derived from polycistronic RNAs. Limited data on operons and spliced leader trans -splicing in other nematodes suggested that SL2-type trans -splicing is a relatively recent innovation, associated with increased efficiency of polycistronic processing, and confined to only one of the five major nematode clades, Clade V. We have conducted the first transcriptome-wide analysis of spliced leader trans -splicing in a nematode species, Trichinella spiralis , which belongs to a clade distantly related to Clade V. Our work identifies a set of T. spiralis SL2-type spliced leaders that are specifically used to process polycistronic RNAs, the first examples of specialised spliced leaders that have been found outside of Clade V. These T. spiralis spliced leader RNAs possess a perfectly conserved stem-loop motif previously shown to be essential for polycistronic processing in C. elegans . We show that this motif is found in specific sets of spliced leader RNAs broadly distributed across the nematode phylum. This work substantially revises our understanding of the evolution of nematode spliced leader trans -splicing, showing that the machinery for SL2 trans -splicing evolved much earlier during nematode evolution than was previously appreciated, and has been conserved throughout the radiation of the nematode phylum.
... The C. elegans genome has a surprisingly varied array of gene clusters. Because the vast majority of such clusters fit the general description of SL2-type operons: ~100 bp intercistronic region containing a Ur element (Lasda et al., 2010) and with almost all trans-splicing to SL2 (Figure 1), it seems likely that this is an optimal arrangement for processing of the polycistronic RNA precursor to produce individual mature mRNAs. Because expression of downstream operon genes depends on successful 3' end formation and trans-splicing, it is not surprising that expression levels of genes more distant from the promoter drop off somewhat (Cutter et al., 2009; M. A. Allen and T. Blumenthal, unpublished observations). ...
Nearly 15% of the ~20,000 C. elegans genes are contained in operons, multigene clusters controlled by a single promoter. The vast majority of these are of a type where the genes in the cluster are ~100 bp apart and the pre-mRNA is processed by 3' end formation accompanied by trans-splicing. A spliced leader, SL2, is specialized for operon processing. Here we summarize current knowledge on several variations on this theme including: (1) hybrid operons, which have additional promoters between genes; (2) operons with exceptionally long (> 1 kb) intercistronic regions; (3) operons with a second 3' end formation site close to the trans-splice site; (4) alternative operons, in which the exons are sometimes spliced as a single gene and sometimes as two genes; (5) SL1-type operons, which use SL1 instead of SL2 to trans-splice and in which there is no intercistronic space; (6) operons that make dicistronic mRNAs; and (7) non-operon gene clusters, in which either two genes use a single exon as the 3' end of one and the 5' end of the next, or the 3' UTR of one gene serves as the outron of the next. Each of these variations is relatively infrequent, but together they show a remarkable variety of tight-linkage gene arrangements in the C. elegans genome.
... This trans-splicing is relatively rare, and is therefore seen more easily with increased numbers of sequencing runs. Many of the less-abundant and newly recognized trans-splicing events mapped to annotated cis-splice sites, a phenomenon recognized previously both in vitro and in vivo (Choi and Newman 2006; Lasda et al. 2010). In fact, because cis-and trans-splice sites share a consensus sequence, it is somewhat surprising that trans-splicing at cis-splice sites is not even more prevalent. ...
Trans-splicing of one of two short leader RNAs, SL1 or SL2, occurs at the 5' ends of pre-mRNAs of many C. elegans genes. We have exploited RNA-sequencing data from the modENCODE project to analyze the transcriptome of C. elegans for patterns of trans-splicing. Transcripts of ∼70% of genes are trans-spliced, similar to earlier estimates based on analysis of far fewer genes. The mRNAs of most trans-spliced genes are spliced to either SL1 or SL2, but most genes are not trans-spliced to both, indicating that SL1 and SL2 trans-splicing use different underlying mechanisms. SL2 trans-splicing occurs in order to separate the products of genes in operons genome wide. Shorter intercistronic distance is associated with greater use of SL2. Finally, increased use of SL1 trans-splicing to downstream operon genes can indicate the presence of an extra promoter in the intercistronic region, creating what has been termed a "hybrid" operon. Within hybrid operons the presence of the two promoters results in the use of the two SL classes: Transcription that originates at the promoter upstream of another gene creates a polycistronic pre-mRNA that receives SL2, whereas transcription that originates at the internal promoter creates transcripts that receive SL1. Overall, our data demonstrate that >17% of all C. elegans genes are in operons.
... In a typical 110-nt ICR, z50 nucleotides (nt) downstream from the 39 cleavage site is a sequence known as the Ur element (Huang et al. 2001; Liu et al. 2003 ). Originally characterized as a z20-nt U-rich sequence, this element has been recently shown to be composed of two distinct components: a stem–loop of variable sequence followed closely by one or more copies of the consensus sequence UAYYUU (Lasda et al. 2010). Both of these features are necessary for SL2 trans-splicing in vitro. ...
... N2 and mutant RNA was isolated and RT-PCR performed as previously described (Lasda et al. 2010 ), using genespecific primers (SupplementalTable S1). PCR products were analyzed by agarose gel electrophoresis. ...
... Since the discovery of operons in C. elegans, they have been characterized primarily by four features: (1) the existence of a single upstream promoter; (2) the close proximity of genes; (3) SL2 trans-splicing of the downstream genes; and (4) the presence of a Ur element within the ICR that is required for SL2 trans-splicing (Spieth et al. 1993; Huang et al. 2001; Lasda et al. 2010). Indeed, these features characterize almost all operons. ...
In Caenorhabditis elegans, the transcripts of many genes are trans-spliced to an SL1 spliced leader, a process that removes the RNA extending from the transcription start site to the trans-splice site, thereby making it difficult to determine the position of the promoter. Here we use RT-PCR to identify promoters of trans-spliced genes. Many genes in C. elegans are organized in operons where genes are closely clustered, typically separated by only ~100 nucleotides, and transcribed by an upstream promoter. The transcripts of downstream genes are trans-spliced to an SL2 spliced leader. The polycistronic precursor RNA is processed into individual transcripts by 3' end formation and trans-splicing. Although the SL2 spliced leader does not appear to be used for other gene arrangements, there is a relatively small number of genes whose transcripts are processed by SL2 but are not close to another gene in the same orientation. Although these genes do not appear to be members of classical C. elegans operons, we investigated whether these might represent unusual operons with long spacing or a different, nonoperon mechanism for specifying SL2 trans-splicing. We show transcription of the entire region between the SL2 trans-spliced gene and the next upstream gene, sometimes several kilobases distant, suggesting that these represent exceptional operons. We also report a second type of atypical "alternative" operon, in which 3' end formation and trans-splicing by SL2 occur within an intron. In this case, the processing sometimes results in a single transcript, and sometimes in two separate mRNAs.
... The C. elegans protocol presented here was used to show SL1 vs SL2 specificity and to determine the characteristics and importance of the Ur element in SL2 trans-splicing. See this reference for examples of some of the different RNA substrates used with the protocol below (Lasda et al. 2010). ...
INTRODUCTION
Caenorhabditis elegans is one of a number of organisms in which spliced leader (SL) trans -splicing contributes to the maturation of many pre-mRNA transcripts. C. elegans and related nematodes have two distinct classes of SL RNA—SL1 and SL2—that are used differentially for different classes of pre-mRNAs; namely, SL1 trans -splices non-operon genes and first genes in operons, while SL2 splices downstream operon genes. In vitro RNA splicing has previously been used to analyze substrate RNA sequences, required protein and RNA factors, and splicing mechanisms in a variety of systems. This protocol describes an in vitro splicing assay using C. elegans extract that promotes both cis - and trans -splicing of substrate RNAs. It has the ability to correctly specify and detect SL1 versus SL2 trans -splicing. Operon-derived substrates splice SL2, while substrates with an intron-like sequence followed by a 3′ trans -splice site (outron) splice SL1. Unlabeled T7-transcribed RNA substrates are incubated with crude embryonic extract and trans -spliced by endogenous SL small nuclear ribonucleoproteins (snRNPs). Spliced RNA is analyzed using a reverse transcriptase polymerase chain reaction (RT-PCR). Specific RT and PCR primers ensure detection of trans -spliced substrate RNA and allow a means to distinguish between SL1 and SL2 trans -spliced products. Substrates containing introns are also cis -spliced. All reaction products are ATP-dependent.
Trans-splicing is a post-transcriptional processing event that joins exons from separate RNAs to produce a chimeric RNA. However, the detailed mechanism of trans-splicing remains poorly understood. Here, we characterize trans-spliced genes and provide insights into the mechanism of trans-splicing in the tunicate Ciona. Tunicates are the closest invertebrates to humans, and their genes frequently undergo trans-splicing. Our analysis revealed that, in genes that give rise to both trans-spliced and non-trans-spliced messenger RNAs, trans-splice acceptor sites were preferentially located at the first functional acceptor site, and their paired donor sites were weak in both Ciona and humans. Additionally, we found that Ciona trans-spliced genes had GU- and AU-rich 5′ transcribed regions. Our data and findings not only are useful for Ciona research community, but may also aid in a better understanding of the trans-splicing mechanism, potentially advancing the development of gene therapy based on trans-splicing.
The human genome is highly dynamic and only a small fraction of it codes for proteins, but most of the genome is transcribed, highlighting the importance of non-coding RNAs on cellular functions. In addition, it is now known the generation of non-coding RNAs fragments under particular cellular conditions and their functions have been revealed unexpected mechanisms of action, converging, in some cases, with the biogenic pathways and action machineries of microRNAs or Piwi-interacting RNAs. This led us to ask why the cell produces so many apparently redundant molecules to exert similar functions and regulate apparently convergent processes? However, non-coding RNAs fragments can also function similarly to aptamers, with secondary and tertiary conformations determining their functions. In the present work, it was reviewed and analyzed the current information about the non-coding RNAs fragments, describing their structure and the biogenic pathways producing them, with special emphasis on their cellular functions.
Spliced leader trans-splicing of pre-mRNAs is a critical step in the gene expression of many eukaryotes. How the spliced leader RNA and its target transcripts are brought together to form the trans-spliceosome remains an important unanswered question. Using immunoprecipitation followed by protein analysis via mass spectrometry and RIP-Seq, we show that the nematode-specific proteins, SNA-3 and SUT-1, form a complex with a set of enigmatic non-coding RNAs, the SmY RNAs. Our work redefines the SmY snRNP and shows for the first time that it is essential for nematode viability and is involved in spliced leader trans-splicing. SNA-3 and SUT-1 are associated with the 5′ ends of most, if not all, nascent capped RNA polymerase II transcripts, and they also interact with components of the major nematode spliced leader (SL1) snRNP. We show that depletion of SNA-3 impairs the co-immunoprecipitation between one of the SL1 snRNP components, SNA-2, and several core spliceosomal proteins. We thus propose that the SmY snRNP recruits the SL1 snRNP to the 5′ ends of nascent pre-mRNAs, an instrumental step in the assembly of the trans-spliceosome.
