The Trypanosoma brucei RNA-binding protein DRBD18 ensures correct mRNA trans splicing and polyadenylation patterns.

The parasite Trypanosoma brucei grows as bloodstream forms in mammals, and as procyclic forms in tsetse flies. Transcription is polycistronic, all mRNAs are trans spliced, and polyadenylation sites are defined by downstream splicing signals. Expression regulation therefore depends heavily on post-transcriptional mechanisms. The RNA-binding protein DRBD18 was previously implicated in the export of some mRNAs from the nucleus in procyclic forms. It copurifies the outer ring of the nuclear pore, mRNA export factors and exon-junction-complex proteins. We show that for more than 200 mRNAs, DRBD18 depletion caused preferential accumulation of versions with shortened 3'-untranslated regions, arising from use of polyadenylation sites that were either undetectable or rarely seen in nondepleted cells. The shortened mRNAs were often, but not always, more abundant in depleted cells than the corresponding longer versions in normal cells. Their appearance was linked to the appearance of trans-spliced, polyadenylated RNAs containing only downstream 3'-untranslated region-derived sequences. Experiments with one mRNA suggested that nuclear retention alone, through depletion of MEX67, did not affect mRNA length, suggesting a specific effect of DRBD18 on processing. DRBD18-bound mRNAs were enriched in polypyrimidine tract motifs, and DRBD18 was found in both the nucleus and the cytoplasm. We therefore suggest that in the nucleus, DRBD18 might bind to polypyrimidine tracts in 3'-UTRs of mRNA precursors. Such binding might both prevent recognition of mRNA-internal polypyrimidine tracts by splicing factors, and promote export of the processed bound mRNAs to the cytosol.

Interactions of the Trypanosoma brucei brucei zinc-finger-domain protein ZC3H28.

In Trypanosoma brucei and related Kinetoplastids, regulation of gene expression occurs mostly post-transcriptionally, and RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Trypanosoma brucei ZC3H28 is a 114 KDa cytoplasmic mRNA-binding protein with a single C(x)7C(x)5C(x)sH zinc finger at the C-terminus and numerous proline-, histidine- or glutamine-rich regions. ZC3H28 is essential for normal bloodstream-form trypanosome growth, and when tethered to a reporter mRNA, ZC3H28 increased reporter mRNA and protein levels. Purification of N-terminally tagged ZC3H28 followed by mass spectrometry showed enrichment of ribosomal proteins, various RNA-binding proteins including both poly(A) binding proteins, the translation initiation complex EIF4E4/EIF4G3, and the activator MKT1. Tagged ZC3H28 was preferentially associated with long RNAs that have low complexity sequences in their 3'-untranslated regions; their coding regions also have low ribosome densities. In agreement with the tethering results, after ZC3H28 depletion, the levels of a significant proportion of its bound mRNAs decreased. We suggest that ZC3H28 is implicated in the stabilization of long mRNAs that are poorly translated.

Several different sequences are implicated in bloodstream-form-specific gene expression in Trypanosoma brucei.

The parasite Trypanosoma brucei grows as bloodstream forms in mammalian hosts, and as procyclic forms in tsetse flies. In trypanosomes, gene expression regulation depends heavily on post-transcriptional mechanisms. Both the RNA-binding protein RBP10 and glycosomal phosphoglycerate kinase PGKC are expressed only in mammalian-infective forms. RBP10 targets procyclic-specific mRNAs for destruction, while PGKC is required for bloodstream-form glycolysis. Developmental regulation of both is essential: expression of either RBP10 or PGKC in procyclic forms inhibits their proliferation. We show that the 3'-untranslated region of the RBP10 mRNA is extraordinarily long-7.3kb-and were able to identify six different sequences, scattered across the untranslated region, which can independently cause bloodstream-form-specific expression. The 3'-untranslated region of the PGKC mRNA, although much shorter, still contains two different regions, of 125 and 153nt, that independently gave developmental regulation. No short consensus sequences were identified that were enriched either within these regulatory regions, or when compared with other mRNAs with similar regulation, suggesting that more than one regulatory RNA-binding protein is important for repression of mRNAs in procyclic forms. We also identified regions, including an AU repeat, that increased expression in bloodstream forms, or suppressed it in both forms. Trypanosome mRNAs that encode RNA-binding proteins often have extremely extended 3'-untranslated regions. We suggest that one function of this might be to act as a fail-safe mechanism to ensure correct regulation even if mRNA processing or expression of trans regulators is defective.