Abstract
We have characterized the RNA-binding protein RBP33 in Trypanosoma brucei, and found that it localizes to the nucleus and is essential for viability. The subset of RNAs bound to RBP33 was determined by immunoprecipitation of ribonucleoprotein complexes followed by deep sequencing. Most RBP33-bound transcripts are predicted to be non-coding. Among these, over one-third are located close to the end of transcriptional units (TUs) or have an antisense orientation within a TU. Depletion of RBP33 resulted in an increase in the level of RNAs derived from regions that are normally silenced, such as strand-switch regions, retroposon and repeat sequences. Our work provides the first example of an RNA-binding protein involved in the regulation of gene silencing in trypanosomes.
Highlights
Trypanosomatids are single-celled eukaryotes that are responsible for major human and livestock diseases
In this work we describe the characterization of the RNAbinding protein RBP33, which was first identified in a search for proteins that were able to bind to a uridine-rich RNA element in vitro [11]
Western blot analysis of total cell extracts using a polyclonal antiserum raised against the carboxyl half of RBP33 [11] revealed a single band with an apparent electrophoretic mobility of 40 kDa, which was expressed at similar levels in the two readily cultured developmental forms, the mammalian bloodstream and the insect procyclic forms (Fig. 1B and Fig. S2A)
Summary
Trypanosomatids are single-celled eukaryotes that are responsible for major human and livestock diseases. Genome organization in these organisms is unusual for a eukaryote, as open reading frames are organized in long polycistronic transcription units (TUs) that are transcribed by RNA polymerase II from only a few initiation sites in the chromosome [1]. RNA polymerase II transcription usually begins at divergent SSRs and ends at convergent SSRs [1,3]. Histone variants present in convergent SSRs are distinct from other variants associated with divergent SSRs, and it is assumed that the limits of polycistronic TUs are defined by distinct chromatin conformations [4]. Chromatin conformation could be important in insulating RNA polymerase II TUs from RNA polymerase I and III expression sites [4,5]
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