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Maturation of polycistronic pre-mRNA in Trypanosoma brucei: analysis of trans splicing and poly(A) addition at nascent RNA transcripts from the hsp70 locus

Numerous protein-coding genes of the protozoan Trypanosoma brucei are arranged in tandem arrays that are transcribed polycistronically. The pre-mRNA transcripts are processed by trans splicing, leading to the addition of a capped 39-nucleotide (nt) miniexon and by poly(A) addition. We wished to determine the order of the RNA processing events at the hsp70 locus and address the potential occurrence of cotranscriptional RNA processing. We determined the rate of transcriptional elongation at the hsp70 locus in isolated nuclei, which measured between 20 and 40 nt/min. This low rate of RNA chain elongation allowed us to label the 3' end of hsp70 nascent RNA with a short (about 180-nt) 32P tail. The structure of the labeled nascent hsp70 RNA could then be analyzed by RNase T1 and RNase T1/RNase A mapping. We show that the trans splicing of hsp70 pre-mRNA did not occur immediately after the synthesis of the 3' splice acceptor site, and nascent RNA molecules that contained about 550 nt of RNA beyond the 3' splice acceptor site still had not acquired a miniexon. In contrast, nascent RNA with a 5' end that mapped to the polyadenylation site of the hsp70 genes could be detected, indicating that maturation of the pre-mRNA in trypanosomes involves a rapid cleavage of the nascent hsp70 RNA (within seconds after synthesis of the site) for poly(A) addition. Our data suggest that polycistronic pre-mRNA is unlikely to be synthesized in toto and rather appears to be processed cotranscriptionally by cleavage for poly(A) addition.