The hnRNP F/H homologue of Trypanosoma brucei is differentially expressed in the two life cycle stages of the parasite and regulates splicing and mRNA stability

Sachin Kumar Gupta, Idit Kosti, Guy Plaut, Asher Pivko, Itai Dov Tkacz, Smadar Cohen-Chalamish, Dipul Kumar Biswas, Chaim Wachtel, Hiba Waldman Ben-Asher, Shai Carmi, Fabian Glaser, Yael Mandel-Gutfreund, Shulamit Michaeli*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

Trypanosomes are protozoan parasites that cycle between a mammalian host (bloodstream form) and an insect host, the Tsetse fly (procyclic stage). In trypanosomes, all mRNAs are trans-spliced as part of their maturation. Genome-wide analysis of trans-splicing indicates the existence of alternative trans-splicing, but little is known regarding RNA-binding proteins that participate in such regulation. In this study, we performed functional analysis of the Trypanosoma brucei heterogeneous nuclear ribonucleoproteins (hnRNP) F/H homologue, a protein known to regulate alternative splicing in metazoa. The hnRNP F/H is highly expressed in the bloodstream form of the parasite, but is also functional in the procyclic form. Transcriptome analyses of RNAi-silenced cells were used to deduce the RNA motif recognized by this protein. A purine rich motif, AAGAA, was enriched in both the regulatory regions flanking the 3′ splice site and poly (A) sites of the regulated genes. The motif was further validated using mini-genes carrying wild-type and mutated sequences in the 3′ and 5′ UTRs, demonstrating the role of hnRNP F/H in mRNA stability and splicing. Biochemical studies confirmed the binding of the protein to this proposed site. The differential expression of the protein and its inverse effects on mRNA level in the two lifecycle stages demonstrate the role of hnRNP F/H in developmental regulation.

Original languageAmerican English
Pages (from-to)6577-6594
Number of pages18
JournalNucleic Acids Research
Volume41
Issue number13
DOIs
StatePublished - Jul 2013
Externally publishedYes

Bibliographical note

Funding Information:
Deutsche Forschungsgemeinschaft via Deutsche-Israelische Projektkooperation and the Israel Science Foundation as well as the I-core grant 41/11. (to S.M. in part). S.M. holds the David and Inez Myers Chair in RNA silencing of diseases. S.C. thanks the Human Frontier Science Program for financial support. Funding for open access charge: The I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation [grant No 41/11].

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