Thomas Blumenthal

Caenorhabditis elegans is unusual among animals in having a highly conserved octamer sequence at the 3′ splice site: UUUU CAG/R. This sequence can bind to the essential heterodimeric splicing factor U2AF, with U2AF⁶⁵ contacting the U tract and U2AF³⁵ contacting the splice site itself (AG/R). Here we demonstrate a strong correspondence between binding to U2AF of RNA oligonucleotides with variant octamer sequences and the frequency with which such variations occur in splice sites. C. elegans U2AF has a strong preference for the octamer sequence and exerts much of the pressure for 3′ splice sites to have the precise UUUUCAG/R sequence. At two positions the splice site has a very strong preference for U even though alternative bases can also bind tightly to U2AF, suggesting that evolution can select against sequences that may have a relatively modest reduction in binding. Although pyrimidines are frequently present at the first base in the exon, U2AF has a very strong bias against them, arguing there is a mechanism to compensate for weakened U2AF binding at this position. Finally, the C in the consensus sequence must remain adjacent to the AG/R rather than to the stretch of U’s, suggesting this C is recognized by U2AF³⁵.

It was thought that polycistronic transcription is a characteristic of bacteria and archaea, where many of the genes are clustered in operons composed of two to more than ten genes. By contrast, the genes of eukaryotes are generally considered to be monocistronic, each with its own promoter at the 5' end and a transcription terminator at the 3' end; however, it has recently become clear that not all eukaryotic genes are transcribed monocistronically. Numerous instances of polycistronic transcription in eukaryotes, from protists to chordates, have been reported. These can be divided into two broad types. Dicistronic transcription units specify a messenger RNA (mRNA) encoding two separate genes that is transported to the cytoplasm and translated in that form. Presumably, internal ribosome entry sites (IRES), or some form of translational re-initiation following the stop codon, are responsible for allowing translation of the downstream gene. In the other type, the initial transcript is processed by 3' end cleavage and trans-splicing to create monocistronic mRNAs that are transported to the cytoplasm and translated. Like bacterial operons, eukaryotic operons often result in co-expression of functionally related proteins.

Expression of a gfp transgene in the intestines of living Caenorhabditis elegans has been measured following depletion by RNAi of a variety of known splicing factors and mRNA export proteins. Reduction of most splicing factors showed only a small effect on expression of the transgene in the animal injected with dsRNA, although most of these RNAi’s resulted in embryonic lethality in their offspring. In contrast, RNAi of nxf-1, the worm homolog of mammalian NXF1/TAP, a key component of the mRNA export machinery, resulted in dramatic suppression of GFP expression in the injected animals. When we tested other proteins previously reported to be involved in marking mRNAs for export, we obtained widely divergent results. Whereas RNAi of the worm REF/Aly homologs had no obvious effect, either in the injected animals or their offspring, RNAi of UAP56, reported to be the partner of REF/Aly, resulted in strong suppression of GFP expression due to nuclear retention of its mRNA. Overexpression of UAP56 also resulted in rapid loss of GFP expression and lethality at all stages of development. We conclude that UAP56 plays a key role in mRNA export in C. elegans, but that REF/Aly may not. It also appears that some RNA processing factors are required for viability (e.g., U2AF, PUF60, SRp54, SAP49, PRP8, U1-70K), whereas others are not (e.g., U2A′, CstF50).

Polycistronic pre-mRNAs from Caenohabditis elegans operons are processed by internal cleavage and polyadenylation to create 3’ ends of mature mRNAs. This is accompanied by trans-splicing with SL2 ∼100 nucleotides downstream of the 3’ end formation sites to create the 5’ ends of downstream mRNAs. SL2 trans-splicing depends on a U-rich element (Ur), located ∼70 nucleotides upstream of the trans-splice site in the intercistronic region (ICR), as well as a functional 3′ end formation signal. Here we report the existence of a novel gene-length RNA, the Ur-RNA, starting just upstream of the Ur element. The expression of Ur-RNA is dependent on 3′ end formation as well as on the presence of the Ur element, but does not require a trans-splice site. The Ur-RNA is not capped, and alteration of the location of the Ur element in either the 5′ or 3′ direction alters the location of the 5′ end of the Ur-RNA. We propose that a 5’ to 3’ exonuclease degrades the precursor RNA following cleavage at the poly(A) site, stopping when it reaches the Ur element, presumably attributable to a bound protein. Part of the function of this protein can be performed by the MS2 coat protein. Recruitment of coat protein to the ICR in the absence of the Ur element results in accumulation of an RNA equivalent to Ur-RNA, and restores trans-splicing. Only SL1, however, is used. Therefore, coat protein is sufficient for blocking the exonuclease and thereby allowing formation of a substrate for trans-splicing, but it lacks the ability to recruit the SL2 snRNP. Our results also demonstrate that MS2 coat protein can be used as an in vivo block to an exonuclease, which should have utility in mRNA stability studies.

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About half of Caenorhabditis elegans genes have a 1–2 bp mismatch to the canonical AAUAAA hexamer that signals 3′ end formation. One rare variant, AGUAAA, is found at the 3′ end of the mai-1 gene, the first gene in an operon also containing gpd-2 and gpd-3. When we expressed this operon under heat shock control, 3′ end formation dependent on the AGUAAA was very inefficient, but could be rescued by a single bp change to create a perfect AAUAAA. When AGUAAA was present, most 3′ ends formed at a different site, 100 bp farther downstream, right at the gpd-2 trans-splice site. Surprisingly, 3′ end formation at this site did not require any observable match to the AAUAAA consensus. It is possible that 3′ end formation at this site occurs by a novel mechanism—trans-splicing-dependent cleavage—as deletion of the trans-splice site prevented 3′ end formation here. Changing the AGUAAA to AAUAAA also influenced the trans-splicing process: with AGUAAA, most of the gpd-2 product was trans-spliced to SL1, rather than SL2, which is normally used at downstream operon trans-splice sites. However, with AAUAAA, SL2 trans-splicing of gpd-2 was increased. Our results imply that (1) the AAUAAA consensus controls 3′ end formation frequency in C. elegans; (2) the AAUAAA is important in determining SL2 trans-splicing events more than 100 bp downstream; and (3) in some circumstances, 3′ end formation may occur by a trans-splicing-dependent mechanism.

In Caenorhabditis elegans, polycistronic pre-mRNAs are processed by cleavage and polyadenylation at the 3' ends of the upstream genes and trans splicing, generally to the specialized spliced leader SL2, at the 5' ends of the downstream genes. Previous studies have indicated a relationship between these two events in the processing of a heat shock-induced gpd-2-gpd-3 polycistronic pre-mRNA. Here, we report mutational analysis of the intercistronic region of this operon by linker scan analysis. Surprisingly, no sequences downstream of the 3' end were important for 3'-end formation. In contrast, a U-rich (Ur) element located 29 bp downstream of the site of 3'-end formation was shown to be important for downstream mRNA biosynthesis. This approximately 20-bp element is sufficient for SL2 trans splicing and mRNA accumulation when transplanted to a heterologous context. Furthermore, when the downstream gene was replaced by a gene from another organism, no loss of trans-splicing specificity was observed, suggesting that the Ur element may be the primary signal required for downstream mRNA processing.

Genes in Caenorhabditis elegans operons are transcribed as polycistronic pre-mRNAs in which downstream gene products aretrans spliced to a specialized spliced leader, SL2. SL2 is donated by a 110-nucleotide RNA, SL2 RNA, present in the cell as an Sm-bound snRNP. SL2 RNA can be conceptually folded into a phylogenetically conserved three-stem-loop secondary structure. Here we report an in vivo mutational analysis of the SL2 RNA. Some sequences can be changed without consequence, while other changes result in a substantial loss of trans splicing. Interestingly, the spliced leader itself can be dramatically altered, such that the first stem-loop cannot form, with only a relatively small loss intrans-splicing efficiency. However, the primary sequence of stem II is crucial for SL2 trans splicing. Similarly, the conserved primary sequence of the third stem-loop plays a key role intrans splicing. While mutations in stem-loop III allow snRNP formation, a single nucleotide substitution in the loop preventstrans splicing. In contrast, the analogous region of SL1 RNA is not highly conserved, and its mutation does not abrogate function. Thus, stem-loop III appears to confer a specific function to SL2 RNA. Finally, an upstream sequence, previously predicted to be a proximal sequence element, is shown to be required for SL2 RNA expression.