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.
Full-textDOI: · Available from: Regina M B Cicarelli, Aug 22, 2015
- SourceAvailable from: Mark Field
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- "This has been coupled with the emergence of RNA interference (RNAi) for suppression of gene expression in a conditional manner, RNA sequencing approaches to monitor transcription (Kolev et al. 2010) and most recently RNAi-based expression knockdown screens (RIT-seq, Alsford et al. 2011), with the result that our understanding of the cell biology and metabolism of T. brucei has advanced at an accelerated pace during the past five to ten years. However, many of the investigations in this period have been centered around 'candidate'-based approaches, i.e. mining the genome for gene products with either known functions or at least functions in known processes or pathways or predictions based on similarity of either sequence or domain architectures; transcription, histone modification, intracellular trafficking and the cytoskeleton are all good examples of where this type of approach has been of great value (see Kawahara et al. 2008; Luz Ambrósio et al. 2009; Field and Carrington, 2009; Wickstead et al. 2010). "
ABSTRACT: Trypanosomatids represent the causative agents of major diseases in humans, livestock and plants, with inevitable suffering and economic hardship as a result. They are also evolutionarily highly divergent organisms, and the many unique aspects of trypanosome biology provide opportunities in terms of identification of drug targets, the challenge of exploiting these putative targets and, at the same time, significant scope for exploration of novel and divergent cell biology. We can estimate from genome sequences that the degree of divergence of trypanosomes from animals and fungi is extreme, with perhaps one third to one half of predicted trypanosome proteins having no known function based on homology or recognizable protein domains/architecture. Two highly important aspects of trypanosome biology are the flagellar pocket and the nuclear envelope, where in silico analysis clearly suggests great potential divergence in the proteome. The flagellar pocket is the sole site of endo- and exocytosis in trypanosomes and plays important roles in immune evasion via variant surface glycoprotein (VSG) trafficking and providing a location for sequestration of various invariant receptors. The trypanosome nuclear envelope has been largely unexplored but, by analogy with higher eukaryotes, roles in the regulation of chromatin and most significantly, in controlling VSG gene expression are expected. Here we discuss recent successful proteomics-based approaches towards characterization of the nuclear envelope and the endocytic apparatus, the identification of conserved and novel trypanosomatid-specific features, and the implications of these findings.Parasitology 02/2012; 139(9):1158-67. DOI:10.1017/S0031182011002125 · 2.35 Impact Factor
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- "Moreover, proteins from the Prp19 complex, some snRNP proteins and other spliceosomal proteins were purified with TAPtagged Cwc21 from yeast, but only Prp8, Prp6 and Brr2 appeared to be U5 snRNP proteins (Grainger et al. 2009, Khanna et al. 2009). The T. brucei U5-Cwc21 description suggests that this protein belongs to the Prp19 complex or the 35S U5 snRNP complex (Luz Ambrósio et al. 2009). Although homologues of hPrp9, CDC5, hSyf1, hIsy1 and Syf3, which are Prp19 complex proteins, have been identified in trypanosomes, proteins that are related to the Prp19 complex were not co-purified with U5-Cwc21-PTP (Günzl 2010). "
ABSTRACT: Several protozoan parasites exist in the Trypanosomatidae family, including various agents of human diseases. Multiple lines of evidence suggest that important differences are present between the translational and mRNA processing (trans splicing) systems of trypanosomatids and other eukaryotes. In this context, certain small complexes of RNA and protein, which are named small nuclear ribonucleoproteins (U snRNPs), have an essential role in pre-mRNA processing, mainly during splicing. Even though they are well defined in mammals, snRNPs are still not well characterized in trypanosomatids. This study shows that a U5-15K protein is highly conserved among various trypanosomatid species. Tandem affinity pull-down assays revealed that this protein interacts with a novel U5-102K protein, which suggests the presence of a sub-complex that is potentially involved in the assembly of U4/U6-U5 tri-snRNPs. Functional analyses showed that U5-15K is essential for cell viability and is somehow involved with the trans and cis splicing machinery. Similar tandem affinity experiments with a trypanonosomatid U5-Cwc21 protein led to the purification of four U5 snRNP specific proteins and a Sm core, suggesting U5-Cwc-21 participation in the 35S U5 snRNP particle. Of these proteins, U5-200K was molecularly characterized. U5-200K has conserved domains, such as the DEAD/DEAH box helicase and Sec63 domains and displays a strong interaction with U5 snRNA.Memórias do Instituto Oswaldo Cruz 03/2011; 106(2):130-8. DOI:10.1590/S0074-02762011000200003 · 1.57 Impact Factor
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- "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. "
ABSTRACT: In Saccharomyces cerevisiae, Cwc21p is a protein of unknown function that is associated with the NineTeen Complex (NTC), a group of proteins involved in activating the spliceosome to promote the pre-mRNA splicing reaction. Here, we show that Cwc21p binds directly to two key splicing factors-namely, Prp8p and Snu114p-and becomes the first NTC-related protein known to dock directly to U5 snRNP proteins. Using a combination of proteomic techniques we show that the N-terminus of Prp8p contains an intramolecular fold that is a Snu114p and Cwc21p interacting domain (SCwid). Cwc21p also binds directly to the C-terminus of Snu114p. Complementary chemical cross-linking experiments reveal reciprocal protein footprints between the interacting Prp8 and Cwc21 proteins, identifying the conserved cwf21 domain in Cwc21p as a Prp8p binding site. Genetic and functional interactions between Cwc21p and Isy1p indicate that they have related functions at or prior to the first catalytic step of splicing, and suggest that Cwc21p functions at the catalytic center of the spliceosome, possibly in response to environmental or metabolic changes. We demonstrate that SRm300, the only SR-related protein known to be at the core of human catalytic spliceosomes, is a functional ortholog of Cwc21p, also interacting directly with Prp8p and Snu114p. Thus, the function of Cwc21p is likely conserved from yeast to humans.RNA 10/2009; 15(12):2161-73. DOI:10.1261/rna.1908309 · 4.62 Impact Factor