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

Abstract

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.

FIGURE 1.

Alignment of Ascaris SL30p with SL21p and SL26p from C. elegans and C. briggsae. Ascaris SL30p sequence is from UniProt (Q8T3T6_ASCSU). C. elegans SL21p and SL26p are encoded by W02F12.6, sna-1, and T13B5.8, sut-1, respectively. The C. briggsae homolog sequences were obtained from Wormbase. The five sequences were aligned using Clustal W. As: Ascaris; br: C. briggsae. Identities between three or more sequences are shown in bold. N-terminal repeats shared by all five proteins are bold underlined, and C-terminal sequences shared only by SL26p are underlined.

FIGURE 2.

The Caenorhabditis Sm Y family. The Sm Y family members were identified using BLAST in both C. elegans (Ce) and C. briggsae (Cb) genomes. Gene names, smy-1–smy-12, and chromosomal locations are given in the Supplemental material available at http://mcdb.colorado.edu/faculty/blumenthal.htm. Sequences were aligned and the dendrogram made by Clustal W using MacVector (UPGMA, best tree). Note that Sm Y-10 is encoded by a distant member of the gene family. The rooting of this tree must be considered uncertain.

FIGURE 3.

Interactions between SL snRNP proteins. (A) SL75p interacts with SL21p and SL26p independent of RNA. Western blot of proteins immunoprecipitated from embryonic extract. Some samples were treated with RNase (+) before IP. The blot was probed with anti-SL26p or anti-SL21p, as indicated on the left. IP antibodies are shown at the top. In each case, input and IP samples shown on the gels are approximately equal percentages of the total. (B) Destruction of snRNAs by RNase 1. Northern blot of RNAs isolated from extract used in panel A. RNAs were detected by hybridization with ³²P-labeled oligonucleotides complementary to SL2 RNA, SL1 RNA, and Sm Y. The Sm Y probe recognizes a number of the Sm Y family members and appears on the Northern blot as a doublet. (Lane 1) control; (lane 2) RNase-treated. (C) SL26p is part of an Sm snRNP. Western blot probed with anti-SL26p antibody. RNase treated (+). IP: lanes 3 and 4, Kung patient serum (anti-Sm); lanes 5 and 6, nonimmune rabbit serum.

FIGURE 4.

Association of SL proteins with snRNAs. (A,C–E) Northern blots of RNA extracted from IPs with antibodies shown at the top, blotted and probed with ³²P-labeled oligonucleotide probes to the snRNAs shown at the left. WT, sut-1 (sut-1 mutant strain). (B) sut-1 mutant lacks SL26p. Western blot of increasing amounts of WT and sut-1 mutant worm proteins, probed with SL26p and SL21p antibodies.

FIGURE 5.

Association of snRNAs and proteins demonstrated by gradient mobility shift assays. Wild-type embryonic extracts were fractionated on 10%–40% glycerol gradients without addition of antibody serum (A) or preincubated with antibodies against SL21p (B) or SL26p (C). Each fraction was divided for RNA and protein analysis. Northern blots were probed for various snRNAs and Western blots probed for SL21p and SL26p. Arrows indicate the approximate sedimentation peak fraction of chicken ovalbumin (∼44 kDa), β amylase (∼200 kDa), and apoferritin (∼443 kDa) run in a parallel gradient.

FIGURE 6.

The Caenorhabditis SL2 RNA genes. The SL2 RNA genes from both Caenorhabdidtis species were identified by BLAST and the alignment and dendrogram produced as in Figure 2. C. elegans (ce); C. briggsae (cb). We have adopted the terminology of Thierry-Mieg (pers. comm.) in which the C. elegans genes are designated SL2–SL12 depending on their frequency in the C. elegans EST database, with SL2 being the most abundant and SL12 the least. Note that these names are dependent on the donated leader sequence, rather than the entire SL RNA sequence, so a single spliced leader is specified by several different genes. For example, SL2 is specified by four genes in C. elegans and eight genes in C. briggsae that contain sequence differences in the SL RNA, but no differences in the spliced leader. In the case of SL3 RNA, identical SLs are specified by distantly related genes (CeSL3-1 closely related to CeSL12; and CeSL3-2 and CeSL3-3 that tree together). The C. briggsae genes are named based on the sequences of their SL, but with two sequences not found in C. elegans called SL13 and SL14. Thus C. briggsae has the following spliced leaders: SL1, SL2, SL3, SL4, SL10, SL13, and SL14.

FIGURE 7.

Possible base pairing between Sm Y and SL RNA family members. The diagram shows a representation of Sm Y (above) and SL RNA (below) with the regions proposed to be involved in base pairing circled. The thickened line shows the SL on the SL RNA, and the box shows the location of the Sm binding site. The proposed C. elegans base pairings are shown on the right, with potential base-paired sequences shown in bold. The loop sequences are underlined. The box shows proposed base pairings of SL1 RNAs with the Sm Y or Sm Y-10 of each species.