EC Accepts, published online ahead of print on 8 May 2009

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Luz Ambrósio, D. Articles by Günzl, A. Search for Related Content PubMed PubMed Citation Articles by Luz Ambrósio, D. Articles by Günzl, A.

 Previous Article  |  Next Article 

Eukaryotic Cell doi:10.1128/EC.00075-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Spliceosomal proteomics in Trypanosoma brucei revealed new RNA splicing factors

Daniela Luz Ambrósio, Ju Huck Lee, Aswini K. Panigrahi, Tu Ngoc Nguyen, Regina Maria Barretto Cicarelli, and Arthur Günzl^*

Department of Genetics and Developmental Biology and Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USA; Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, SP, Brazil; Seattle Biomedical Research Institute, Seattle, WA, USA

^* To whom correspondence should be addressed. Email: gunzl{at}uchc.edu.

In trypanosomatid parasites, spliced leader (SL) trans splicing is an essential nuclear mRNA maturation step which caps mRNAs post-transcriptionally 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 co-purified proteins by mass spectrometry. The set of 47 identified proteins harbored nearly all spliceosomal snRNP factors characterized in trypanosomes so far and twenty-one proteins lacking a specific annotation. A bioinformatic analysis combined with protein pull-down assays and immunofluorescence microscopy identified nine divergent orthologues of known splicing factors including the missing U1-specific protein U1A. In addition, a novel U5-specific, and as we show, essential splicing factor was identified which shares a short, highly conserved N-terminal domain with the yeast protein Cwc21p and thus was tentatively named U5-Cwc21. Together, these data strongly indicate that most of the identified proteins are components of the spliceosome.