Spliceosomal Proteomics in Trypanosoma brucei Reveal New RNA Splicing Factors

Abstract

In trypanosomatid parasites, spliced leader (SL) trans splicing is an essential nuclear mRNA maturation step which caps mRNAs posttranscriptionally and, in conjunction with polyadenylation, resolves individual mRNAs from polycistronic precursors. While all trypanosomatid mRNAs are trans spliced, intron removal by cis splicing is extremely rare and predicted to occur in only four pre-mRNAs. trans-and cis-splicing reactions are carried out by the spliceosome, which consists of U-rich small nuclear ribonucleoprotein particles (U snRNPs) and of non-snRNP factors. Mammalian and yeast spliceosome complexes are well characterized and found to be associated with up to 170 proteins. Despite the central importance of trans splicing in trypanosomatid gene expression, only the core RNP proteins and a few snRNP-specific proteins are known. To characterize the trypanosome spliceosomal protein repertoire, we conducted a proteomic analysis by tagging and tandem affinity-purifying the canonical core RNP protein SmD1 in Trypanosoma brucei and by identifying copurified proteins by mass spectrometry. The set of 47 identified proteins harbored nearly all spliceosomal snRNP factors characterized in trypanosomes thus far and 21 proteins lacking a specific annotation. A bioinformatic analysis combined with protein pull-down assays and immunofluorescence microscopy identified 10 divergent orthologues of known splicing factors, including the missing U1-specific protein U1A. In addition, a novel U5-specific, and, as we show, an essential splicing factor was identified that shares a short, highly conserved N-terminal domain with the yeast protein Cwc21p and was thus tentatively named U5-Cwc21. Together, these data strongly indicate that most of the identified proteins are components of the spliceosome.Trypanosomatid parasites utilize RNA splicing for the maturation of nuclear pre-mRNA in two distinct ways: first, as in other eukaryotes, cis splicing is used for intron removal. While this was unambiguously demonstrated for the Trypanosoma brucei poly(A) polymerase (PAP) mRNA (19), intron removal appears to be a rare event in trypanosomatids because a survey of the Tritryp genomes has identified only three additional putative intron-containing genes (10). Second, trypanosomatids process all of their nuclear pre-mRNAs by spliced leader (SL) trans splicing (reviewed in reference 15). In this splicing reaction, the capped, 39 nucleotide (nt)-long 5Ј terminus of the SL RNA, the SL or miniexon, is fused to the 5Ј end of each mRNA. This process is therefore a posttranscriptional mRNA capping mechanism and, since trypanosomatids transcribe their protein coding genes polycistronically, it resolves individual mRNAs from polycistronic precursors in conjunction with polyadenylation. SL trans splicing occurs in several organisms, including tunicate chordates, nematodes, and trematodes, but it is not found in insect and mammalian cells; thus, it is specific to the parasites and not present in the hosts of trypanosomatids. Hence, factors with specific fun...

Physical and genetic interactions of yeast Cwc21p, an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome

“…The sequence similarities between Cwc21p and the N terminus of the human SRm300 have been noted previously (Blencowe et al 2000;Luz Ambrosio et al 2009). We confirm here that the N-terminal 159 amino acids of human SRm300 can functionally substitute for yeast Cwc21p in vivo and that both bind directly to the yeast Prp8p SCwid via a conserved region called the cwf21 domain.…”

“…As an NTC or NTC-related protein, Cwc21p is in a position to function within the spliceosome B* and C complexes during the stages of spliceosome activation and catalysis. Cwc21p is a short protein of 135 residues whose primary sequence has been aligned with the N terminus of human SRm300 (encoded by the SRRM2 gene) (Blencowe et al 2000;Luz Ambrosio et al 2009) and they are thought to be orthologs, although this has not been confirmed experimentally. SRm300 was first isolated through its stringent copurification with SRm160 (Blencowe et al 1995;Blencowe et al 1998).…”

Tools for Trans-Splicing Drug Interference Evaluation in Kinetoplastid

“…Cwc21 protein, which was described being exclusive of trypanosomatids, proved to be essential for survival of the parasite and its silencing caused inference in both cis-and trans-splicing (Ambrósio et al, 2009). Several other examples are presented in the literature as highly conserved protein of U5 snRNP, U5-15K (da and recently described SMN (Palfi et al, 2009) from T. brucei, whose participation in splicing was detected using semi-quantitative RT-PCR.…”

“…PremRNA (pre-α Tub) and mature mRNA of α-tubulin (α-Tub) were amplified in the fourth and fifth panels, respectively. The figure 8 B was transcribed from Ambrósio et al (2009).…”

The polyadenylation complex ofTrypanosoma brucei:Characterization of the functional poly(A) polymerase

“…Although studies in the last decade have identified numerous spliceosomal components, we still know very little about the composition and functioning of the polyadenylation machinery in trypanosomes. 1,2,3 In higher eukaryotes the polyadenylation complex consists of a multitude of proteins, which assemble in an orchestrated manner on their target RNAs. The specificity of 3 0 end processing of newly transcribed RNA is driven by two cis-acting elements: the highly conserved AAUAAA hexanucleotide, located 10-30 nucleotides upstream of the pre-mRNA cleavage site, and a U-or GU-rich sequence around 30 nucleotides downstream of the cleavage site.…”