Different trans RNA splicing events in bloodstream and procyclic Trypanosoma brucei

Abstract

A distinctive feature of Trypanosoma brucei and other trypanosomatids is that most of their genes are transcribed into long polycistronic RNAs [1], which are processed into mature monocistronic mRNAs bearing a 39-nucleotide spliced leader (SL) at their 5′ ends that is derived from a capped precursor RNA of ∼140 nucleotides [2;3]. This trans RNA splicing serves two major functions: in conjunction with 3′ polyadenylation it generates the mature mRNAs from polycistronic primary transcripts, and via the SL it provides a 5′ cap structure for each mRNA [1;4]. In contrast to cis RNA splicing of introns in other eukaryotes, trans RNA splicing unites exons from two independently transcribed RNAs. trans and cis RNA splicing, however, share several similarities. For example, at least some components of the cis RNA spliceosomes are conserved in T. brucei, and both cis and trans RNA splicing utilize the same general mechanism and require the same general sequence motifs [4]. During an examination of the effect of different 3′ UTRs on gene expression in T. brucei, we noticed differences between bloodstream form (BSF) and procyclic form (PCF) trypanosomes in the addition of SLs to RNAs transcribed from a luciferase reporter gene inserted into the rRNA gene locus. Further inspection revealed that in PCF cells the expected SL addition occurs to precursor luciferase RNA, whereas in BSF cells multiple SL additions occur to the same luciferase RNA that are independent of the 5′ and 3′ UTR sequences. Fig. 1A depicts the luciferase reporter plasmid, pMP, integrated into the rRNA gene spacer region of the T. brucei genome that was used for the experiments. This plasmid is derived from plasmid pHD496 (kindly provided by C. Clayton), and is used to measure luciferase activity in BSF and PCF cells when various 3′-UTRs are inserted at a BamHI site directly behind the luciferase coding sequence (LUC). An initial northern blot of RNA from BSF cells bearing integrated pMP revealed the presence of multiple RNA species recognized by a LUC probe (Fig 1B). The three bands indicated by arrowheads coincide with the locations of rRNAs and are likely due to entrapment of some luciferase RNA in these rRNAs. The RNA bands indicated by L (long), LUC and S (short) were examined further. To look for potential differential luciferase expression from the same inserted plasmid in BSF and PCF cells of the same * Corresponding author. Tel.: +1-319-335-7934; fax: +1-319-335-9570, Email address: E-mail: john-donelson@uiowa.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH...

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