Polycistronic pre-mRNAs Research Articles

Structural basis of Spliced Leader RNA recognition by the Trypanosoma brucei cap-binding complex

Kinetoplastids are a clade of eukaryotic protozoans that include human parasitic pathogens like trypanosomes and Leishmania species. In these organisms, protein-coding genes are transcribed as polycistronic pre-mRNAs, which need to be processed by the coupled action of trans-splicing and polyadenylation to yield monogenic mature mRNAs. During trans-splicing, a universal RNA sequence, the spliced leader RNA (SL RNA) mini-exon, is added to the 5’-end of each mRNA. The 5’-end of this mini-exon carries a hypermethylated cap structure and is bound by a trypanosomatid-specific cap-binding complex (CBC). The function of three of the kinetoplastid CBC subunits is unknown, but an essential role in cap-binding and trans-splicing has been suggested. Here, we report cryo-EM structures that reveal the molecular architecture of the Trypanosoma brucei CBC (TbCBC) complex. We find that TbCBC interacts with two distinct features of the SL RNA. The TbCBP20 subunit interacts with the m7G cap while TbCBP66 recognizes double-stranded portions of the SL RNA. Our findings pave the way for future research on mRNA maturation in kinetoplastids. Moreover, the observed structural similarities and differences between TbCBC and the mammalian cap-binding complex will be crucial for considering the potential of TbCBC as a target for anti-trypanosomatid drug development.

SLIDR and SLOPPR: flexible identification of spliced leader trans-splicing and prediction of eukaryotic operons from RNA-Seq data

BackgroundSpliced leader (SL) trans-splicing replaces the 5′ end of pre-mRNAs with the spliced leader, an exon derived from a specialised non-coding RNA originating from elsewhere in the genome. This process is essential for resolving polycistronic pre-mRNAs produced by eukaryotic operons into monocistronic transcripts. SL trans-splicing and operons may have independently evolved multiple times throughout Eukarya, yet our understanding of these phenomena is limited to only a few well-characterised organisms, most notably C. elegans and trypanosomes. The primary barrier to systematic discovery and characterisation of SL trans-splicing and operons is the lack of computational tools for exploiting the surge of transcriptomic and genomic resources for a wide range of eukaryotes.ResultsHere we present two novel pipelines that automate the discovery of SLs and the prediction of operons in eukaryotic genomes from RNA-Seq data. SLIDR assembles putative SLs from 5′ read tails present after read alignment to a reference genome or transcriptome, which are then verified by interrogating corresponding SL RNA genes for sequence motifs expected in bona fide SL RNA molecules. SLOPPR identifies RNA-Seq reads that contain a given 5′ SL sequence, quantifies genome-wide SL trans-splicing events and predicts operons via distinct patterns of SL trans-splicing events across adjacent genes. We tested both pipelines with organisms known to carry out SL trans-splicing and organise their genes into operons, and demonstrate that (1) SLIDR correctly detects expected SLs and often discovers novel SL variants; (2) SLOPPR correctly identifies functionally specialised SLs, correctly predicts known operons and detects plausible novel operons.ConclusionsSLIDR and SLOPPR are flexible tools that will accelerate research into the evolutionary dynamics of SL trans-splicing and operons throughout Eukarya and improve gene discovery and annotation for a wide range of eukaryotic genomes. Both pipelines are implemented in Bash and R and are built upon readily available software commonly installed on most bioinformatics servers. Biological insight can be gleaned even from sparse, low-coverage datasets, implying that an untapped wealth of information can be retrieved from existing RNA-Seq datasets as well as from novel full-isoform sequencing protocols as they become more widely available.

Computational analysis of spliced leader trans-splicing in the regenerative flatworm Macrostomum lignano reveals its prevalence in conserved and stem cell related genes.

In eukaryotes, trans-splicing is a process of nuclear pre-mRNA maturation where two different RNA molecules are joined together by the spliceosomal machinery utilizing mechanisms similar to cis-splicing. In diverse taxa of lower eukaryotes, spliced leader (SL) trans-splicing is the most frequent type of trans-splicing, when the same sequence derived from short small nuclear RNA molecules, called SL RNAs, is attached to the 5’ ends of different non-processed pre-mRNAs. One of the functions of SL trans-splicing is processing polycistronic pre-mRNA molecules transcribed from operons, when several genes are transcribed as one pre-mRNA molecule. However, only a fraction of trans-spliced genes reside in operons, suggesting that SL trans-splicing must also have some other, less understood functions. Regenerative flatworms are informative model organisms which hold the keys to understand the mechanism of stem cell regulation and specialization during regeneration and homeostasis. Their ability to regenerate is fueled by the division and differentiation of the adult somatic stem cell population called neoblasts. Macrostomum lignano is a flatworm model organism where substantial technological advances have been achieved in recent years, including the development of transgenesis. Although a large fraction of genes in M. lignano were estimated to be SL trans-spliced, SL trans-splicing was not studied in detail in M. lignano before. Here, we performed the first comprehensive study of SL trans-splicing in M. lignano. By reanalyzing the existing genome and transcriptome data of M. lignano, we estimate that 30 % of its genes are SL trans-spliced, 15 % are organized in operons, and almost 40 % are both SL trans-spliced and in operons. We annotated and characterized the sequence of SL RNA and characterized conserved cis- and SL transsplicing motifs. Finally, we found that a majority of SL trans-spliced genes are evolutionarily conserved and significantly over-represented in neoblast-specific genes. Our findings suggest an important role of SL trans-splicing in the regulation and maintenance of neoblasts in M. lignano.

A Trypanosoma cruzi zinc finger protein that is implicated in the control of epimastigote-specific gene expression and metacyclogenesis.

Trypanosoma cruzi has three biochemically and morphologically distinct developmental stages that are programmed to rapidly respond to environmental changes the parasite faces during its life cycle. Unlike other eukaryotes, Trypanosomatid genomes contain protein coding genes that are transcribed into polycistronic pre-mRNAs and have their expression controlled by post-transcriptional mechanisms. Transcriptome analyses comparing three stages of the T. cruzi life cycle revealed changes in gene expression that reflect the parasite adaptation to distinct environments. Several genes encoding RNA binding proteins (RBPs), known to act as key post-transcriptional regulatory factors, were also differentially expressed. We characterized one T. cruzi RBP, named TcZH3H12, which contains a zinc finger domain and is up-regulated in epimastigotes compared to trypomastigotes and amastigotes. TcZC3H12 knockout (KO) epimastigotes showed decreased growth rates and increased capacity to differentiate into metacyclic trypomastigotes. Transcriptome analyses comparing wild type and TcZC3H12 KOs revealed a TcZC3H12-dependent expression of epimastigote-specific genes such as genes encoding amino acid transporters and proteins associated with differentiation (PADs). RNA immunoprecipitation assays showed that transcripts from the PAD family interact with TcZC3H12. Taken together, these findings suggest that TcZC3H12 positively regulates the expression of genes involved in epimastigote proliferation and also acts as a negative regulator of metacyclogenesis.

The Role of E6 Spliced Isoforms (E6*) in Human Papillomavirus-Induced Carcinogenesis.

Persistent infections with High Risk Human Papillomaviruses (HR-HPVs) are the main cause of cervical cancer development. The E6 and E7 oncoproteins of HR-HPVs are derived from a polycistronic pre-mRNA transcribed from an HPV early promoter. Through alternative splicing, this pre-mRNA produces a variety of E6 spliced transcripts termed E6*. In pre-malignant lesions and HPV-related cancers, different E6/E6* transcriptional patterns have been found, although they have not been clearly associated to cancer development. Moreover, there is a controversy about the participation of E6* proteins in cancer progression. This review addresses the regulation of E6 splicing and the different functions that have been found for E6* proteins, as well as their possible role in HPV-induced carcinogenesis.

Serine/Arginine-Rich Splicing Factor 3 and Heterogeneous Nuclear Ribonucleoprotein A1 Regulate Alternative RNA Splicing and Gene Expression of Human Papillomavirus 18 through Two Functionally Distinguishable cis Elements.

Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA splicing remain unknown. In this study, an exonic splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 splicing and E1^E4 production through interaction with SRSF3, a host oncogenic splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An exonic splicing silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting splicing factors. Expression of HPV18 genes is regulated by alternative RNA splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 splicing in the E6 ORF through interaction with a host splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host splicing factors and offers potential therapeutic targets to overcome HPV-related cancer.

Operons are a conserved feature of nematode genomes.

The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla. Operons are present in the class Chromadorea, one of the two main nematode classes, but their distribution in the other class, the Enoplea, is not known. We have surveyed the genomes of Trichinella spiralis, Trichuris muris, and Romanomermis culicivorax and identified the first putative operons in members of the Enoplea. Consistent with the mechanism of polycistronic RNA resolution in other nematodes, the mRNAs produced by genes downstream of the first gene in the T. spiralis and T. muris operons are trans-spliced to spliced leader RNAs, and we are able to detect polycistronic RNAs derived from these operons. Importantly, a putative intercistronic region from one of these potential enoplean operons confers polycistronic processing activity when expressed as part of a chimeric operon in Caenorhabditis elegans. We find that T. spiralis genes located in operons have an increased likelihood of having operonic C. elegans homologs. However, operon structure in terms of synteny and gene content is not tightly conserved between the two taxa, consistent with models of operon evolution. We have nevertheless identified putative operons conserved between Enoplea and Chromadorea. Our data suggest that operons and “spliced leader” (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing.

Trans-splicing and operons in C. elegans.

About 70% of C. elegans mRNAs are trans-spliced to one of two 22 nucleotide spliced leaders. SL1 is used to trim off the 5' ends of pre-mRNAs and replace them with the SL1 sequence. This processing event is very closely related to cis-splicing, or intron removal. The SL1 sequence is donated by a 100 nt small nuclear ribonucleoprotein particle (snRNP), the SL1 snRNP. This snRNP is structurally and functionally similar to the U snRNAs (U1, U2, U4, U5 and U6) that play key roles in intron removal and trans-splicing, except that the SL1 snRNP is consumed in the process. More than half of C. elegans pre-mRNAs are subject to SL1 trans-splicing, whereas ~30% are not trans-spliced. The remaining genes are trans-spliced by SL2, which is donated by a similar snRNP, the SL2 snRNP. SL2 recipients are all downstream genes in closely spaced gene clusters similar to bacterial operons. They are transcribed from a promoter at the 5' end of the cluster of between 2 and 8 genes. This transcription makes a polycistronic pre-mRNA that is co-transcriptionally processed by cleavage and polyadenylation at the 3' end of each gene, and this event is closely coupled to the SL2 trans-splicing event that occurs only ~100 nt further downstream. SL2 trans-splicing requires a sequence between the genes, the Ur element, that likely base pairs with the 5' splice site on the SL2 snRNP, in a manner analogous to the interaction between the 5' splice site in cis-splicing with the U1 snRNP. The key difference is that in trans-splicing, the snRNP contains the 5' splice site, whereas in cis-splicing the pre-mRNA does. Some operons, termed "hybrid operons", contain an additional promoter between two genes that can express the downstream gene or genes with a developmental profile that is different from that of the entire operon. The operons contain primarily genes required for rapid growth, including genes whose products are needed for mitochondrial function and the basic machinery of gene expression. Recent evidence suggests that RNA polymerase is poised at the promoters of growth genes, and operons allow more efficient recovery from growth-arrested states, resulting in reduction in the need for this cache of inactive RNA polymerase.

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5'ETS molecule using three distinct methods and located the acceptor site between two known 5'ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5'ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5'ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5'ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

A global analysis of C. elegans trans-splicing

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.

Polycistronic pre-mRNA processing in vitro: snRNP and pre-mRNA role reversal in trans-splicing

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, and that trans-splicing specificity is recapitulated in vitro. We define an RNA sequence required for SL2 trans-splicing, the U-rich (Ur) element, through mutational analysis and bioinformatics as a short stem-loop followed by a sequence motif, UAYYUU, located approximately 50 nt upstream of the trans-splice site. Furthermore, this element is predicted in intercistronic regions of numerous operons of C. elegans and other species that use SL2 trans-splicing. We propose that the UAYYUU motif hybridizes with the 5' splice site on the SL2 RNA to recruit the SL to the pre-mRNA. In this way, the UAYYUU motif in the pre-mRNA would serve an analogous function to the similar sequence in the U1 snRNA, which binds to the 5' splice site of introns, effectively reversing the roles of snRNP and pre-mRNA in trans-splicing.

Alternative splicing of human papillomavirus type-16 E6/E6* early mRNA is coupled to EGF signaling via Erk1/2 activation

Certain types of human papillomaviruses (HPVs) are etiologically linked to cervical cancer. Their transforming capacity is encoded by a polycistronic premRNA, where alternative splicing leads to the translation of functional distinct proteins such as E6, E6*, and E7. Here we show that splicing of HPV16 E6/E7 ORF cassette is regulated by the epidermal growth factor (EGF) pathway. The presence of EGF was coupled to preferential E6 expression, whereas depletion of EGF, or treatment with EGF receptor (EGFR) neutralizing antibodies or the EGFR inhibitor tyrphostin AG1478, resulted in E6 exon exclusion in favor of E6*. As a consequence, increased p53 levels and enhanced translation of E7 with a subsequent reduction of the retinoblastoma protein pRb could be discerned. E6 exon exclusion upon EGF depletion was independent from promoter usage, mRNA stability, or selective mRNA transport. Time-course experiments and incubation with cycloheximide demonstrated that E6 alternative splicing is a direct and reversible effect of EGF signal transduction, not depending on de novo protein synthesis. Within this process, Erk1/2-kinase activation was the critical event for E6 exon inclusion, mediated by the upstream MAP kinase MEK1/2. Moreover, siRNA knockdown experiments revealed an involvement of splicing factors hnRNPA1 and hnRNPA2 in E6 exon exclusion, whereas the splicing factors Brm and Sam68 were found to promote E6 exon inclusion. Because there is a natural gradient of EGF and EGF receptor expression in the stratified epithelium, it is reasonable to assume that EGF modulates E6/E7 splicing during the viral life cycle and transformation.

Regulation of transcription termination in the nematode Caenorhabditis elegans

The current predicted mechanisms that describe RNA polymerase II (pol II) transcription termination downstream of protein expressing genes fail to adequately explain, how premature termination is prevented in eukaryotes that possess operon-like structures. Here we address this issue by analysing transcription termination at the end of single protein expressing genes and genes located within operons in the nematode Caenorhabditis elegans. By using a combination of RT-PCR and ChIP analysis we found that pol II generally transcribes up to 1 kb past the poly(A) sites into the 3′ flanking regions of the nematode genes before it terminates. We also show that pol II does not terminate after transcription of internal poly(A) sites in operons. We provide experimental evidence that five randomly chosen C. elegans operons are transcribed as polycistronic pre-mRNAs. Furthermore, we show that cis-splicing of the first intron located in downstream positioned genes in these polycistronic pre-mRNAs is critical for their expression and may play a role in preventing premature pol II transcription termination.

Special Sm Core Complex Functions in Assembly of the U2 Small Nuclear Ribonucleoprotein of Trypanosoma brucei

The processing of polycistronic pre-mRNAs in trypanosomes requires the spliceosomal small ribonucleoprotein complexes (snRNPs) U1, U2, U4/U6, U5, and SL, each of which contains a core of seven Sm proteins. Recently we reported the first evidence for a core variation in spliceosomal snRNPs; specifically, in the trypanosome U2 snRNP, two of the canonical Sm proteins, SmB and SmD3, are replaced by two U2-specific Sm proteins, Sm15K and Sm16.5K. Here we identify the U2-specific, nuclear-localized U2B'' protein from Trypanosoma brucei. U2B'' interacts with a second U2 snRNP protein, U2-40K (U2A'), which in turn contacts the U2-specific Sm16.5K/15K subcomplex. Together they form a high-affinity, U2-specific binding complex. This trypanosome-specific assembly differs from the mammalian system and provides a functional role for the Sm core variation found in the trypanosomal U2 snRNP.

Leishmania ( Viannia) braziliensis transfectants overexpressing the miniexon gene lose virulence in vivo

The miniexon gene has a central role in the processing of polycistronic pre-mRNA of kinetoplastids. It is added to the 5′ extremity of each mRNA, supplying the 5′-capped structure to the molecule. Previous studies in Leishmania ( Leishmania) major showed that the overexpression of the miniexon array attenuates the virulence of the parasite in in vivo assays. The results presented here extend those findings to Viannia subgenus. Leishmania ( Viannia) braziliensis was transfected with a cosmid harboring a tandem array of one hundred miniexon gene copies and then characterized by Northern blot analysis. The overexpression of the exogenous gene was confirmed and its effect on the virulence of L. ( V.) braziliensis was investigated in hamsters. In BALB/c mice we could not detect parasites during the course of 15 weeks of infection. In addition, hamsters infected with transfectants overexpressing the miniexon gene exhibited only a minor footpad swelling of late onset and failed to develop progressive lesion, these attenuated parasites could be recovered from the inoculation site 1 year after infection. The persistence of parasites in the host indicates that a stable line overexpressing the miniexon may be tested as live vaccine against leishmaniasis.

C. elegans sequences that control trans-splicing and operon pre-mRNA processing.

Many mRNAs in Caenorhabditis elegans are generated through a trans-splicing reaction that adds one of two classes of spliced leader RNA to an independently transcribed pre-mRNA. SL1 leaders are spliced mostly to pre-mRNAs from genes with outrons, intron-like sequences at the 5'-ends of the pre-mRNAs. In contrast, SL2 leaders are nearly exclusively trans-spliced to genes that occur downstream in polycistronic pre-mRNAs produced from operons. Operon pre-mRNA processing requires separation into individual transcripts, which is accomplished by 3'-processing of upstream genes and spliced leader trans-splicing to the downstream genes. We used a novel computational analysis, based on nonnegative matrix factorization, to identify and characterize significant differences in the cis-acting sequence elements that differentiate various types of functional site, including internal versus terminal 3'-processing sites, and SL1 versus SL2 trans-splicing sites. We describe several key elements, including the U-rich (Ur) element that couples 3'-processing with SL2 trans-splicing, and a novel outron (Ou) element that occurs upstream of SL1 trans-splicing sites. Finally, we present models of the distinct classes of trans-splicing reaction, including SL1 trans-splicing at the outron, SL2 trans-splicing in standard operons, competitive SL1-SL2 trans-splicing in operons with large intergenic separation, and SL1 trans-splicing in SL1-type operons, which have no intergenic separation.

A novel family of C. elegans snRNPs contains proteins associated with trans-splicing.

In many Caenorhabditis elegans pre-mRNAs, the RNA sequence between the 5' cap and the first 3' splice site is replaced by trans-splicing a short spliced leader (SL) from the Sm snRNP, SL1. C. elegans also utilizes a similar Sm snRNP, SL2, to trans-splice at sites between genes in polycistronic pre-mRNAs from operons. How do SL1 and SL2 snRNPs function in different contexts? Here we show that the SL1 snRNP contains a complex of SL75p and SL21p, which are homologs of novel proteins previously reported in the Ascaris SL snRNP. Interestingly, we show that the SL2 snRNP does not contain these proteins. However, SL75p and SL26p, a paralog of SL21p, are components of another Sm snRNP that contains a novel snRNA species, Sm Y. Knockdown of SL75p is lethal. However, knockdown of either SL21p or SL26p alone leads to cold-sensitive sterility, whereas knockdown of both SL21p and SL26p is lethal. This suggests that these two proteins have overlapping functions even though they are associated with different classes of snRNP. These phenotypic relationships, along with the association of SL26p with SL75p, imply that, like the SL1 RNA/Sm/SL75p/SL21p complex, the Sm Y/Sm/SL75p/SL26p complex is associated with trans-splicing.

Operon Conservation and the Evolution of trans-Splicing in the Phylum Nematoda

The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five major clades of the phylum Nematoda, for the presence of operons and the use of trans-spliced leaders in resolution of polycistronic pre-mRNAs. Conserved operons were found in Pristionchus pacificus, Nippostrongylus brasiliensis, Strongyloides ratti, Brugia malayi, and Ascaris suum. In nematodes closely related to the rhabditine C. elegans, a related family of SL2-like spliced leaders is used for operonic transcript resolution. However, in the tylenchine S. ratti operonic transcripts are resolved using a family of spliced leaders related to SL1. Non-operonic genes in S. ratti may also receive these SL1 variants. In the spirurine nematodes B. malayi and A. suum operonic transcripts are resolved using SL1. Mapping these phenotypes onto the robust molecular phylogeny for the Nematoda suggests that operons evolved before SL2-like spliced leaders, which are an evolutionary invention of the rhabditine lineage.

Trans-splicing and operons

About 70% of C. elegans mRNAs are trans-spliced to one of two 22 nucleotide spliced leaders. SL1 is used to trim off the 5' ends of pre-mRNAs and replace them with the SL1 sequence. This processing event is very closely related to cis-splicing, or intron removal. The SL1 sequence is donated by a 100 nt small nuclear ribonucleoprotein particle (snRNP). This snRNP is structurally and functionally related to the U snRNAs (U1, U2, U4, U5 and U6) that play key roles in intron removal and trans-splicing, except that it is consumed in the process of splicing. More than half of C. elegans pre-mRNAs are subject to SL1 trans-splicing. About 30% are not trans-spliced at all. The remaining genes are trans-spliced by SL2. These genes are all downstream genes in closely spaced gene clusters similar to bacterial operons. They are transcribed from a promoter at the 5' end of the cluster of between 2 and 8 genes. This transcription makes a polycistronic pre-mRNA that is co-transcriptionally processed by cleavage and polyadenylation at the 3' end of each gene, and this event is closely coupled to the SL2 trans-splicing event that occurs only approximately 100 nt further downstream. Recent studies on the mechanism of SL2 trans-splicing have revealed that one of the 3' end formation proteins, CstF, interacts with the only protein known to be specific to the SL2 snRNP. The operons contain primarily genes whose products are needed for mitochondrial function and the basic machinery of gene expression: transcription, splicing and translation. Many operons contain genes whose products are known to function together. This presumably provides co-regulation of these proteins by producing a single RNA that encodes both.

Spliced-leader RNA trans splicing in a chordate, Oikopleura dioica, with a compact genome.

trans splicing of a spliced-leader RNA (SL RNA) to the 5' ends of mRNAs has been shown to have a limited and sporadic distribution among eukaryotes. Within metazoans, only nematodes are known to process polycistronic pre-mRNAs, produced from operon units of transcription, into mature monocistronic mRNAs via an SL RNA trans-splicing mechanism. Here we demonstrate that a chordate with a highly compact genome, Oikopleura dioica, now joins Caenorhabditis elegans in coupling trans splicing with processing of polycistronic transcipts. We identified a single SL RNA which associates with Sm proteins and has a trimethyl guanosine cap structure reminiscent of spliceosomal snRNPs. The same SL RNA, estimated to be trans-spliced to at least 25% of O. dioica mRNAs, is used for the processing of both isolated or first cistrons and downstream cistrons in a polycistronic precursor. Remarkably, intercistronic regions in O. dioica are far more reduced than those in either nematodes or kinetoplastids, implying minimal cis-regulatory elements for coupling of 3'-end formation and trans splicing.