3' Untranslated regions regulate developmental expression of two RNA binding proteins, p34 and p37, from Trypanosoma brucei
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Two homologous proteins, p34 and p37, are developmentally regulated in Trypanosoma brucei , with p34 primarily expressed in procyclic form and p37 exclusively expressed in bloodstream form. Previous studies have shown that the mRNA stability of p34 is decreased in bloodstream form, while the translation efficiency and protein stability of p37 are decreased in procyclic form. The transcripts of p34 and p37 contain divergent sequences in their 3' untranslated regions. Using reporter gene analysis, we have shown that the p34 3' UTR decreased reporter mRNA stability in bloodstream form. Deletion of a 17-nt AU rich element (ARE) from the p34 3' UTR enhanced the mRNA stability. The ARE itself, when inserted into a control 3' UTR sequence, did not regulate gene expression. Secondary structure analysis and RNase sensitivity assays showed that the 17-nt ARE is located in a single-stranded region in the p34 3' UTR. Deletion of this ARE decreased the size of the single-stranded region and the sensitivity to RNase A. The reporter gene analysis also showed that the p37 3' UTR developmentally regulates translation efficiency. Deletion of a 29-nt polypyrimidine sequence from the p37 3' UTR did not change the expression of reporter luciferase. However, insertion of the 29-nt sequence in control 3' UTR decreased the expression of reporter luciferase in both life stages of T. brucei. Secondary structure analysis indicated that deletion of the 29-nt sequence did not change the structure of the p37 3' UTR, which forms a long stem structure with nearly all of the 3' UTR sequences residing in a double stranded region. The insertion of the 29-nt sequence alone moderately modified the structure of the control 3' UTR, causing a short U rich sequence to reside in a single stranded region. All these results indicate that the secondary structures are involved in gene regulation controlled by the 3' UTRs of p34 and p37. We have identified proteins that bind to the p34 and/or p37 3' UTR. T. brucei HMG like TDP-1 protein binds to both 3' UTRs without poly (A) tails, while addition of a poly (A) tail inhibited binding of TDP-1 to the p37 3' UTR. TDP-1 binds to the mRNAs of p34 and the T. brucei phosphoglycerate kinase (PGKB) gene but not to the p37 mRNA. Both PGKB and p34 are regulated at the mRNA stability level by AREs in the 3' UTRs. These results indicated that the TDP-1 protein is involved in regulation of mRNA stability. In vitro RNA degradation assays have shown that TDP-1 protected RNA with the p34 3' UTR sequences from degradation in T. brucei bloodstream cell extracts. A second protein, glycerol kinase (GLK) from T. brucei was shown to bind RNAs with U rich sequences. The GLK protein showed higher affinity to the p37 3' UTR compared to the p34 3' UTR.