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.

Two splicing factors carrying serine-arginine motifs, TSR1 and TSR1IP, regulate splicing, mRNA stability, and rRNA processing inTrypanosoma brucei

“…In addition, the protein recognition site, AAGAA, is located around the 3′ splice site and the poly (A) site. 60 In this study, we found that substrates downregulated by TSR1 and TSR1IP silencing contain weaker splicing signals such as shorter and pyrimidinepoor PPTs, and that the regulated genes have more heterogeneous poly (A) site selection. RNA-seq of these transcriptomes may reveal additional substrates, especially non-abundant transcripts that are not detected by the microarray analysis but also have weak splicing signals, and hence, rely on SR proteins for their splicing.…”

The proteome and transcriptome of the infectious metacyclic form of Trypanosoma brucei define quiescent cells primed for mammalian invasion

“…Our data provide further evidence supporting the important role of post‐transcriptional gene‐expression regulators in controlling T. brucei development. Among factors with previously demonstrated regulatory activity are the RNA‐binding proteins RBP10 and hnRNP F/H, two proteins with clear functions in establishing a bloodstream‐form type transcriptome (Wurst et al ., ; Gupta et al ., ; De Pablos et al ., ), which are highly up‐regulated in metacyclic trypanosomes (Fig. A).…”

“…Among factors with previously demonstrated regulatory activity are the RNA-binding proteins RBP10 and hnRNP F/H, two proteins with clear functions in establishing a bloodstream-form type transcriptome (Wurst et al, 2012;Gupta et al, 2013;De Pablos et al, 2017), which are highly up-regulated in metacyclic trypanosomes (Fig. 5A).…”

“…RBP10 mRNA was increased fivefold in metacyclics. HnRNP F/H (Tb927.2.3880), the first pre-mRNA transsplicing regulator shown to be differentially expressed in African trypanosomes, also has a role in the cytoplasm by affecting mRNA stability (Gupta et al, 2013) and is up-regulated in metacyclics fivefold at the protein level ( Fig. 5A).…”

Depletion of the Trypanosome Pumilio Domain Protein PUF2 or of Some Other Essential Proteins Causes Transcriptome Changes Related to Coding Region Length

“…Two life cycle stages are routinely cultured in the laboratory: the bloodstream form, which is similar to the forms that multiply in mammalian blood and tissue fluids, and the procyclic form, which is similar to the parasites in the tsetse fly midgut. Different mRNA levels-and also regulation during differentiation-are achieved by modulation of mRNA decay (17) and trans-splicing (18)(19)(20). Trypanosome mRNA decay pathways are similar to those of other eukaryotes: mRNAs are deadenylated by a CAF1-NOT complex (21), then the mRNA body is attacked from the 3= end by the exosome (22)(23)(24) and in the 5=-3= direction by XRNA (25).…”