Genome-wide analysis of mRNA abundance in two life-cycle stages of Trypanosoma brucei and identification of splicing and polyadenylation sites

Abstract

Transcription of protein-coding genes in trypanosomes is polycistronic and gene expression is primarily regulated by post-transcriptional mechanisms. Sequence motifs in the untranslated regions regulate mRNA trans-splicing and RNA stability, yet where UTRs begin and end is known for very few genes. We used high-throughput RNA-sequencing to determine the genome-wide steady-state mRNA levels (‘transcriptomes’) for ∼90% of the genome in two stages of the Trypanosoma brucei life cycle cultured in vitro. Almost 6% of genes were differentially expressed between the two life-cycle stages. We identified 5′ splice-acceptor sites (SAS) and polyadenylation sites (PAS) for 6959 and 5948 genes, respectively. Most genes have between one and three alternative SAS, but PAS are more dispersed. For 488 genes, SAS were identified downstream of the originally assigned initiator ATG, so a subsequent in-frame ATG presumably designates the start of the true coding sequence. In some cases, alternative SAS would give rise to mRNAs encoding proteins with different N-terminal sequences. We could identify the introns in two genes known to contain them, but found no additional genes with introns. Our study demonstrates the usefulness of the RNA-seq technology to study the transcriptional landscape of an organism whose genome has not been fully annotated.

Insights into the regulation of GPEET procyclin during differentiation from early to late procyclic forms of Trypanosoma brucei

“…5B). These minor poly(A) sites were also reported in previous studies using RNA-Seq technology [42,43].…”

“…Furthermore, the most frequent oligo(U) tail was enriched in late PF cultures 6 h after addition of ActD. Why did transcriptomewide analyses previously performed in T. brucei [42,43] not identify the uridylyl tails? One possibility is that the mapping algorithms employed resulted in annotation of the sequences as non-assignable reads and might have been ignored.…”

Regulatory elements involved in the post-transcriptional control of stage-specific gene expression in Trypanosoma cruzi: a review

“…While no sequence consensus for polyadenylation or SL addition have been found in trypanosomatids, several studies have demonstrated that polypyrimidine-rich regions within intergenic regions guide SL addition and the polyadenylation of upstream and downstream genes, respectively, resulting in the generation of mature mRNAs (Hartmann et al 1998, López-Estraño et al 1998. The sequence requirements for T. cruzi, T. brucei and Leishmania mRNA processing were initially investigated by comparing expressed sequence tags and/or cDNAs with genomic sequences (Benz et al 2005, Campos et al 2008, Smith et al 2008 and, more recently, they have been investigated using deep sequencing RNA (Kolev et al 2010, Nilsson et al 2010, Siegel et al 2010. Campos et al (2008) reported the average distance from the polypyrimidine tract to the SL addition site and to the polyadenylation site as well as the median lengths of the 5' and 3' untranslated regions (UTR) in T. cruzi genes.…”

The polyadenylation complex ofTrypanosoma brucei:Characterization of the functional poly(A) polymerase

“…34 Since alternative polyadenylation is common in trypanosomes as well, CFIm25 identified here is a potential regulator of this process. 35,36 In other eukaryotes, a direct linkage between spliceosomal and 3 0 end processing components had been demonstrated by several studies. [37][38][39] In trypanosomes, so far only the polypyrimidine tract is known to affect trans splicing and polyadenylation of two adjacent genes.…”