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 structure and trans-splicing in the nematode Pristionchus pacificus

In the nematode Caenorhabditis elegans, up to 15% of the genes are organized in operons. Polycistronic precursor RNAs are processed by trans-splicing at the 5' ends of genes by adding a specific trans-spliced leader. Ten different spliced leaders are known in C. elegans that differ in sequence and abundance. The SL1 leader is most abundant and is spliced to the 5' ends of monocistronic genes and to upstream genes in operons. Trans-splicing is common among nematodes and was observed in the genera Panagrellus, Ascaris, Haemonchus, Anisakis, and Brugia. However, little is known about operons in nonrhabditid nematodes. Dolichorhabditis CEW1, another rhabditid nematode that is now called Oscheius CEW1, contains operons and SL2 trans-splicing. We have studied the presence of operons and trans-splicing in Pristionchus pacificus, a species of the Diplogastridae that has recently been developed as a satellite organism in evolutionary developmental biology. We provide evidence that P. pacificus contains operons and that downstream genes are trans-spliced to SL2. Surprisingly, the one operon analyzed so far in P. pacificus is not conserved in C. elegans, suggesting unexpected genomic plasticity.     

Diversity of 7 SL RNA from the signal recognition particle of maize endosperm.

An 11 S ribonucleoprotein particle was isolated from maize endosperm and shown to be functionally and structurally equivalent to the mammalian signal recognition particle. However, unlike animal cells which apparently contain a single 7 SL RNA species, maize endosperm contains a heterogeneous population of 7 SL RNA. To investigate this diversity, we have cloned and sequenced a number of the maize endosperm 7 SL RNAs isolated from functionally active SRP preparations. Some maize 7 SL RNAs are strikingly similar, differing by single base changes or short deletions; surprisingly, others share less than 70 percent sequence homology. Despite differences in primary sequence, nearly identical secondary structures can be suggested for all maize 7 SL RNAs, consistent with a proposed functional role in protein translocation for each of these RNAs. The amount of new available sequence data enabled us to define two conserved regions of presumed functional importance: A conserved sequence -G-N-A-R- in the center of a variable region which forms a well defined stem-loop and possibly is involved in an interaction with the 19 kDa protein of the SRP. Secondly, three short nucleotide stretches located in the central domain of 7 SL RNA may form part of a dynamic RNA-switch structure.     

Trans splicing of nematode pre-messenger RNA in vitro

In nematodes, a fraction of mRNAs contains a common 22 nucleotide 5' terminal spliced leader (SL) sequence derived by trans splicing. Here, we show that a cell-free extract prepared from developing embryos of the parasitic nematode Ascaris lumbricoides catalyzes accurate and efficient SL addition to a synthetic pre-mRNA at an authentic trans splice acceptor site. SL addition occurs via a trans splicing reaction that proceeds through Y-branched intermediates. The branchpoint is located at either of two adenosine residues located 18 and 19 nucleotides upstream of the splice acceptor site.     

SL1 trans-splicing specified by AU-rich synthetic RNA inserted at the 5' end of Caenorhabditis elegans pre-mRNA.

In Caenorhabditis elegans, pre-mRNAs of many genes are trans-spliced to one of two spliced leaders, SL1 or SL2. Some of those that receive exclusively SL1 have been characterized as having at their 5' ends outrons, AU-rich sequences similar to introns followed by conventional 3' splice sites. Comparison of outrons from many different SL1-specific C. elegans genes has not revealed the presence of any consensus sequence that might encode SL1-specificity. In order to determine what parameters influence the splicing of SL1, we performed in vivo experiments with synthetic splice sites. Synthetic AU-rich RNA, 51 nt or longer, placed upstream of a consensus 3' splice site resulted in efficient trans-splicing. With all sequences tested, this trans-splicing was specifically to SL1. Thus, no information beyond the presence of AU-rich RNA at least as long as the minimum-length C. elegans intron, followed by a 3' splice site, is required to specify trans-splicing or for strict SL1 specificity.     

Most mRNAs in the nematode Ascaris lumbricoides are trans-spliced: a role for spliced leader addition in translational efficiency.

Some pre-mRNAs in nematodes are processed by trans-splicing. In this reaction, a 22-nt 5' terminal exon (the spliced leader, SL) and its associated 2,2,7-trimethylguanosine cap are acquired from a specialized Sm snRNP, the SL RNP. Although it has been evident for many years that not all nematode mRNAs contain the SL sequence, the prevalence of trans-spliced mRNAs has, with the exception of Caenorhabditis elegans, not been determined. To address this question in an organism amenable to biochemical analysis, we have prepared a message-dependent protein synthesis system from developing embryos of the parasitic nematode, Ascaris lumbricoides. Using this system, we have used both hybrid-arrest and hybrid-selection approaches to show that the vast majority (80-90%) of A. lumbricoides mRNAs contain the SL sequence and therefore are processed by trans-splicing. Furthermore, to examine the effect of SL addition on translation, we have measured levels of protein synthesis in extracts programmed with a variety of synthetic mRNAs. We find that the SL sequence itself and its associated hypermethylated cap functionally collaborate to enhance translational efficiency, presumably at the level of initiation of protein synthesis. These results indicate that trans-splicing plays a larger role in nematode gene expression than previously suspected.     

Cell proteins bind specifically to West Nile virus minus-strand 3' stem-loop RNA.

The first 96 nucleotides of the 5'noncoding region (NCR) of West Nile virus (WNV) genomic RNA were previously reported to form thermodynamically predicted stem-loop (SL) structures that are conserved among flaviviruses. The complementary minus-strand 3' NCR RNA, which is thought to function as a promoter for the synthesis of plus-strand RNA, forms a corresponding predicted SL structure. RNase probing of the WNV 3' minus-strand stem-loop RNA [WNV (-)3' SL RNA] confirmed the existence of a terminal secondary structure. RNA-protein binding studies were performed with BHK S100 cytoplasmic extracts and in vitro-synthesized WNV (-)3' SL RNA as the probe. Three RNA-protein complexes (complexes 1,2, and 3) were detected by a gel mobility shift assay, and the specificity of the RNA-protein interactions was confirmed by gel mobility shift and UV-induced cross-linking competition assays. Four BHK cell proteins with molecular masses of 108, 60, 50, and 42 kDa were detected by UV-induced cross-linking to the WNV (-)3' SL RNA. A preliminary mapping study indicated that all four proteins bound to the first 75 nucleotides of the WNV 3' minus-strand RNA, the region that contains the terminal SL. A flavivirus resistance phenotype was previously shown to be inherited in mice as a single, autosomal dominant allele. The efficiencies of infection of resistant cells and susceptible cells are similar, but resistant cells (C3H/RV) produce less genomic RNA than congenic, susceptible cells (C3H/He). Three RNA-protein complexes and four UV-induced cross-linked cell proteins with mobilities identical to those detected in BHK cell extracts with the WNV (-)3' SL RNA were found in both C3H/RV and C3H/He cell extracts. However, the half-life of the C3H/RV complex 1 was three times longer than that of the C3H/He complex 1. It is possible that the increased binding activity of one of the resistant cell proteins for the flavivirus minus-strand RNA could result in a reduced synthesis of plus-strand RNA as observed with the flavivirus resistance phenotype.     

trans splicing of polycistronic Caenorhabditis elegans pre-mRNAs: analysis of the SL2 RNA

Genes in Caenorhabditis elegans operons are transcribed as polycistronic pre-mRNAs in which downstream gene products are trans 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 in trans-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 in trans splicing. While mutations in stem-loop III allow snRNP formation, a single nucleotide substitution in the loop prevents trans 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.     

Intramolecular base pairing between the nematode spliced leader and its 5'' splice site is not essential for trans-splicing in vitro.

The spliced leader RNAs of both trypanosomes and nematodes can form similar secondary structures where the trans-splice donor site is involved in intramolecular base pairing with the spliced leader sequence. It has been proposed that this base pairing could serve to activate autonomously the SL RNA splice donor site. Here, we have examined exon requirements for trans-splicing in a nematode cell free system. Complete disruption of secondary structure interactions at and around the trans-splice donor site did not affect the ability of the SL RNA to function in trans-splicing. In addition, the highly conserved 22 nt sequence could be productively replaced by artificial exons ranging in size from 2 to 246 nucleotides. These results reinforce the view that the 'intron' portion of the SL RNA functions as an independent Sm snRNP whose role is to deliver exon sequences to the trans-spliceosome.     

Structural basis for nematode eIF4E binding an m(2,2,7)G-Cap and its implications for translation initiation

Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m^2,2,7G-cap and a common downstream SL RNA sequence. The mechanism for eIF4E binding an m^2,2,7G-cap is unknown. Here, we describe the first structure of an eIF4E with an m^2,2,7G-cap and compare it to the cognate m⁷G-eIF4E complex. These structures and Nuclear Magnetic Resonance (NMR) data indicate that the nematode Ascaris suum eIF4E binds the two different caps in a similar manner except for the loss of a single hydrogen bond on binding the m^2,2,7G-cap. Nematode and mammalian eIF4E both have a low affinity for m^2,2,7G-cap compared with the m⁷G-cap. Nematode eIF4E binding to the m⁷G-cap, m^2,2,7G-cap and the m^2,2,7G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m^2,2,7G-SL. We propose interactions between Ascaris eIF4E and the SL impact eIF4G and contribute to translation initiation, whereas these interactions do not occur when only the m^2,2,7G-cap is present. These data have implications for the contribution of 5′-UTRs in mRNA translation and the function of different eIF4E isoforms.