The 5' end structure of transcripts derived from the rRNA gene and the RNA polymerase I transcribed protein coding genes in Trypanosoma brucei.

Abstract

Due to trans-splicing and polycistronic transcription, the 5' end structure of precursor RNAs of protein coding genes in Trypanosoma brucei has not yet been characterized. In eukaryotes, in general, the 5' ends of transcripts generated by RNA polymerase (pol) I and pol II are different. Pol I derived precursor RNAs contain an unmodified tri- or diphosphate group at their 5' ends. In contrast, pol II primary transcripts, the 5' triphosphate (initially also part of the pre-mRNA) is rapidly modified by the addition of methylated guanosine triphosphate, immediately after transcription initiation. We determined the 5' end structure of precursor RNAs of the rRNA gene and the RNA pol I transcribed protein coding gene by the differential display of RNA ligase mediated amplification of cDNA ends (DDRLACE) method. Comparing the ability of the 5' end of RNA transcripts to ligate with an RNA primer following different pre-treatments, the structure of the 5' end of RNA transcripts was characterized. We found that: (1). the 5' end of putative precursor RNAs from a pol I transcribed protein coding gene and the rRNA gene was uncapped; (2). approximately 20% of the putative rRNA precursor contained a 5' tri- or diphosphate group, representing the primary transcript and approximately 80% of the putative rRNA precursor were dephosphorylated and contained a 5' hydroxyl group; (3). the majority of putative neomycin resistance gene precursor RNAs, driven by the procyclin gene promoter (a pol I promoter), contained a 5' hydroxyl group. The procyclin-neo primary transcript, as being those containing a 5' tri- or diphosphate, was below a detectable level in the steady state RNA; and (4). we did not detect pol I transcribed precursor RNAs that contained a 5' monophosphate group. The observation that the putative pre-RNAs derived from the procyclin gene promoter, similar to those of rRNA do not have a 5' capped structure, is consistent with the notion that transcription of pol I transcribed protein coding genes is crucially dependent on trans-splicing for the cap addition.