Sequences and proteins that influence mRNA processing in Trypanosoma brucei: evolutionary conservation of SR-domain and PTB protein functions

Abstract

Spliced leader trans splicing is the addition of a short, capped sequence to the 5’ end of mRNAs. It is widespread in eukaryotic evolution, but factors that influence trans splicing acceptor site choice have been little investigated. In Kinetoplastids, all protein-coding mRNAs are 5’ trans spliced. A polypyrimidine tract is usually found upstream of the AG splice acceptor, but there is no branch point consensus; moreover, splicing dictates polyadenylation of the preceding mRNA. We here describe a trans splicing reporter system that can be used for studies and screens concerning the roles of sequences and proteins in processing site choice and efficiency. Splicing was poor with poly(U) tracts less than 9 nt long, and was influenced by nearby secondary structures. A screen for signals resulted in selection of sequences that were on average 45% U and 35% C. Tethering of either the splicing factor SF1, or the cleavage and polyadenylation factor CPSF3 within the intron stimulated processing in the correct positions, while tethering of two possible homologues of Opisthokont PTB inhibited processing. In contrast, tethering of SR-domain proteins RBSR1, RBSR2, or TSR1 or its interaction partner TSR1IP, promoted use of alternative signals upstream of the tethering sites. RBSR1 interacts predominantly with proteins implicated in splicing, whereas the interactome of RBSR2 is more diverse. These results suggest that the functions of PTB and SR-domain proteins in splice site definition were already present in the last eukaryotic common ancestor.

The Trypanosoma brucei RNA-binding protein DRBD18 ensures correct mRNA trans splicing and polyadenylation patterns

“…ZC3H28 is an RNA-binding protein that is predominantly in the cytoplasm ( Bishola Tshitenge and Clayton 2021 ). In addition, we compared the list of specifically associated proteins with that from a separate analysis of the putative splicing factor RBSR1 ( Waithaka et al 2022 ), because it is a nuclear protein ( Supplemental Table S2 , sheet 1).…”

“…By analogy with results from animal cells, DRBD18 activity at wild-type splice acceptor sites may be inhibited by proteins that show higher-affinity binding and specifically define exons. There is evidence for mRNA processing regulation by several nuclear trypanosome RNA-binding proteins (Stern et al 2009;Gupta et al 2013a;Gupta et al 2013b;Gupta et al 2014;Waithaka et al 2022). Further analysis of these and other nuclear RNA-binding proteins will be needed to understand how sites for trypanosome RNA processing are normally determined.…”

“…In trypanosomes, the only splicing signal that has been investigated in detail is the PPT. Results from one set of reporter experiments indicated that maximal splicing efficiency was obtained with PPTs of (U) 20 or with 17mers of poly(U) containing interpellated C residues ( Siegel et al 2005 ), but another series indicated that a 10mer PPT interrupted by a single “A” was as good as a 20mer ( Matthews et al 1994 ), and results of a recent screen suggest even more flexibility ( Waithaka et al 2022 ). In practice, most mRNAs are trans -spliced downstream from PPTs that are 12–24 nt long, with more U than C ( Kolev et al 2010 ); but for some acceptors the PPT is barely recognizable.…”

“…Like other eukaryotes, trypanosomes have an exon junction complex, which is presumably deposited at the end of the spliced leader after or during processing: it includes Y14 (Tb927.7.1170), Mago-Nashi (Tb927.6.4950), and an NTF2-like protein (Tb927.10.2240) ( Bercovich et al 2009 ) and perhaps an eIF4AIII homolog, Tb927.11.8770 ( Dhalia et al 2006 ; Inoue et al 2014 ). Bioinformatic analysis and reporter experiments indicate that—as in other eukaryotes—a polypyrimidine tract (PPT) serves as the main trans -splicing signal ( Matthews et al 1994 ; Schürch et al 1994 ; Vassella et al 1994 ; Nilsson et al 2010 ; Siegel et al 2010 ; Waithaka et al 2022 ), but in contrast with at least some eukaryotes ( Patzelt et al 1989 ), there is no consensus branch-point sequence. The results of reporter experiments indicate that the nature of the sequence downstream from the splice site, as well as the length and composition of the PPT, influence splice site choice ( Hartmann et al 1998 ; Lopez-Estrano et al 1998 ; Siegel et al 2005 ; Waithaka et al 2022 ).…”

“…Bioinformatic analysis and reporter experiments indicate that—as in other eukaryotes—a polypyrimidine tract (PPT) serves as the main trans -splicing signal ( Matthews et al 1994 ; Schürch et al 1994 ; Vassella et al 1994 ; Nilsson et al 2010 ; Siegel et al 2010 ; Waithaka et al 2022 ), but in contrast with at least some eukaryotes ( Patzelt et al 1989 ), there is no consensus branch-point sequence. The results of reporter experiments indicate that the nature of the sequence downstream from the splice site, as well as the length and composition of the PPT, influence splice site choice ( Hartmann et al 1998 ; Lopez-Estrano et al 1998 ; Siegel et al 2005 ; Waithaka et al 2022 ). There are also indications that some mRNAs are more efficiently spliced than others ( Kapotas and Bellofatto 1993 ; Fadda et al 2014 ; Antwi et al 2016 ); and when processing is inhibited, the precursors are substrates for the exosome ( Kramer et al 2016 ).…”