In trypanosomes, mRNAs are processed bytrans-splicing; in this process, a common exon, the spliced leader, is added to all mRNAs from a small RNA donor, the spliced leader RNA (SL RNA). However, little is known regarding how this process is regulated. In this study, we investigated the function of two serine-arginine-rich proteins, TSR1 and TSR1IP, implicated intrans-splicing inTrypanosoma brucei. Depletion of these factors by RNAi suggested their role in bothcis- andtrans-splicing. Microarray was used to examine the transcriptome of the silenced cells. The level of hundreds of mRNAs was changed, suggesting that these proteins have a role in regulating only a subset ofT. bruceimRNAs. Mass-spectrometry analyses of complexes associated with these proteins suggest that these factors function in mRNA stability, translation, and rRNA processing. We further demonstrate changes in the stability of mRNA as a result of depletion of the two TSR proteins. In addition, rRNA defects were observed under the depletion of U2AF35, TSR1, and TSR1IP, but not SF1, suggesting involvement of SR proteins in rRNA processing.