title: 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.
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.
authors.affiliation: Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
authors.forename: Sachin Kumar
authors.initials: SK
authors.lastname: Gupta
authors.forename: Idit
authors.initials: I
authors.lastname: Kosti
authors.forename: Guy
authors.initials: G
authors.lastname: Plaut
authors.forename: Asher
authors.initials: A
authors.lastname: Pivko
authors.forename: Itai Dov
authors.initials: ID
authors.lastname: Tkacz
authors.forename: Smadar
authors.initials: S
authors.lastname: Cohen-Chalamish
authors.forename: Dipul Kumar
authors.initials: DK
authors.lastname: Biswas
authors.forename: Chaim
authors.initials: C
authors.lastname: Wachtel
authors.forename: Hiba
authors.initials: H
authors.lastname: Waldman Ben-Asher
authors.forename: Shai
authors.initials: S
authors.lastname: Carmi
authors.forename: Fabian
authors.initials: F
authors.lastname: Glaser
authors.forename: Yael
authors.initials: Y
authors.lastname: Mandel-Gutfreund
authors.forename: Shulamit
authors.initials: S
authors.lastname: Michaeli
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008018:Life Cycle Stages; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D020040:Trans-Splicing; D059467:Transcriptome; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D015800:Protozoan Proteins; D012333:RNA, Messenger