Spliceosomal Proteomics in Trypanosoma brucei Reveal New RNA Splicing Factors

Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3301, USA.
Eukaryotic Cell (Impact Factor: 3.18). 06/2009; 8(7):990-1000. DOI: 10.1128/EC.00075-09
Source: PubMed


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.

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Available from: Regina M B Cicarelli
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    • "Although these are likely the only two cis-introns in T. brucei, this explains the existence of a U1 snRNP in trypanosomes: cis-splicing requires the recognition of the 5′ splice sites through base-pairing between the U1 snRNA and the 5′ splice site on the pre-mRNA (4). The trypanosome U1 snRNP is unusual in several aspects: its three specific protein components, U1-70K, U1C and U1A, are only distantly related to their known counterparts from other eukaryotes; in addition, U1-24K was characterized as a trypanosomatid-specific U1 snRNP protein, which is stably integrated into the U1 snRNP by protein–protein interactions (5,6). Interestingly, the trypanosome U1 snRNA with 75 nucleotides represents one of the smallest known snRNAs, and lacks a stem-loop II element, which in other orthologs contains the well-characterized U1A binding site. "
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    ABSTRACT: Trans-splicing in trypanosomes adds a 39-nucleotide mini-exon from the spliced leader (SL) RNA to the 5′ end of each protein-coding sequence. On the other hand, cis-splicing of the few intron-containing genes requires the U1 small nuclear ribonucleoprotein (snRNP) particle. To search for potential new functions of the U1 snRNP in Trypanosoma brucei, we applied genome-wide individual-nucleotide resolution crosslinking-immunoprecipitation (iCLIP), focusing on the U1 snRNP-specific proteins U1C and U1-70K. Surprisingly, U1C and U1-70K interact not only with the U1, but also with U6 and SL RNAs. In addition, mapping of crosslinks to the cis-spliced PAP [poly(A) polymerase] pre-mRNA indicate an active role of these proteins in 5′ splice site recognition. In sum, our results demonstrate that the iCLIP approach provides insight into stable and transient RNA–protein contacts within the spliceosomal network. We propose that the U1 snRNP may represent an evolutionary link between the cis- and trans-splicing machineries, playing a dual role in 5′ splice site recognition on the trans-spliceosomal SL RNP as well as on pre-mRNA cis-introns.
    Full-text · Article · Apr 2014 · Nucleic Acids Research
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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). "
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    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.
    Full-text · Article · Feb 2012 · Parasitology
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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). "
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    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.
    Full-text · Article · Mar 2011 · Memórias do Instituto Oswaldo Cruz
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