1. Splicing regulation in Trypanosoma brucei Sachin Kumar Gupta, Shai Carmi, Asher Pivko, Ilana Naboishchikov, Mark Katzenellenbogen, and Shulamit Michaeli The Mina and Everard Goodman Faculty of Life Sciences and Advanced materials & Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel U2AF35 SF1 Depletion of splicing factors affects splicing  Trypanosoma brucei is a unicellular blood parasite that diverged early from the eukaryotic lineage and is the causative agent of the devastating African Sleeping Sickness.  Messenger RNA metabolism in this parasite is unique: genes are transcribed as polycistronic units and are processed by trans-splicing and polyadenylation.  No transcriptional regulation exists in T. brucei. Introduction PTB trans-splicing Several basal splicing factors were identified in T. brucei including factors known to bind the pre-mRNA such as U2AF65, SF1, U2AF35, SR and hnRNP proteins. U2AF65 Fig 2: (a) The trypanosome U2AF65 has the conserved N-terminal RS basic domain, followed by a low complexity region. The C-terminal portion contains three RRM domains (I, II and III) arranged in tandem. (b) U2AF35 lacks tryptophan residue at position 134. (c) SF1 lacks the C-terminal pro-rich region and CCHC domain but contains the conserved KH domain. (d) PTB proteins in trypanosome resemble their mammalian homologues. 1.Is trans-splicing a regulated process? 2.What is the role of trans-splicing in determining the transcriptome? 3.What is the molecular mechanism of trans-splicing regulation? Stable cell lines expressing inducible RNAi constructs were prepared for U2AF65, U2AF35, SF1, TSR1 and TSR1IP. RNA was extracted from silenced cells and hybridized to T. brucei microarray. Genes that were down- or up-regulated by at least 1.5 fold were listed and categorized to their different biological functions. Results Depletion of splicing factors differentially affects the transcriptome Many genes are upregulated; each splicing factor has distinct effect. Ribosomal and ribosome biogenesis proteins are upregulated upon splicing factor silencing. Mechanism is mRNA stability. A. Mammalian cis-splicing B. Tryp trans-splicing C. PTB dependent trans-splicing Intron 65 RRM RRM RRM 3 WF W 35 W SF1 Exon YNYURACUUUUUUUUNYAG Very StrongStrong Intron 65 RRM RR M3 W ORF UUUAUUUUUUUUUUUUUU-(4-40nt)---AG WeakStrong SF1 35 KL 65 RRM RR M3 W ORF UUCUUCUUCUUCCC-------------AG SF1 35 (15 to 40nt, 25nt optimum) spacer (20nt optimum) PTB1 17nt A PTB proteins differentially regulate splicing of genes carrying C-rich polypyrimidine tract. Conclusions Splicing factors contribute to transcriptome regulation in T. brucei. Regulation is specific- each factor affects a different set of genes. Surprisingly, silencing of splicing factors led to upregulation of several transcripts mediated by mRNA stabilization. Downregulation of other transcripts may be due to either inhibition of trans-splicing or destabilization. Ribosomal and ribosome related proteins are upregulated upon silencing, whereas metabolic proteins and transporters are downregulated. Experiments are in progress to map the basal factors binding sites in the 3’UTRs of the regulated genes using bioinformatics and in the future using deep-sequencing. Fig 1: Schematic presentation of the trans-splicing reaction. In trans-splicing, a small exon (spliced leader, SL RNA) is donated to the mRNA. In trypanosomes, every mRNA is trans-spliced. Fig 3: (a) Primer extensions with antisense oligo of spliced leader (SL) RNA was used to determine splicing defects. The results show that U2AF65, U2AF35, and SF1 silencing affect the first step of trans-splicing, while Tsr1 and Tsr1IP silencing affect the second step. (b) A heat-map showing the differential effect of the splicing factors on the transcriptome. Fig 4: Effect of U2AF65 and SF1 silencing on mRNA stability. The half-life of selected mRNAs was calculated in control and silenced cells after Actinomycin D treatment. The results demonstrate that upregulated mRNA are stabilized during silencing. Fig 5: In trypanosomes, U2AF65 and U2AF35 don’t interact. C-rich polypyrimidine tracts require either PTB1 or PTB2 for their splicing. PTB1 interact with U2AF65 in an RNA-independent manner. a b cd ab Research questions: Research strategy: