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Hierarchies of RNA-processing signals in a trypanosome surface antigen mRNA precursor
1Zentrum für Molekulare Biologie, Heidelberg, Federal Republic of Germany.
Abstract:
Nearly all trypanosome mRNAs are synthesized as polycistronic precursors, from which mature mRNAs are excised by trans splicing and polyadenylation. Polyadenylation of a procyclic acidic repetitive protein (PARP, or procyclin) transcript was studied by transient transfection of constructs bearing a chloramphenicol acetyltransferase gene linked to the PARP intergenic region. Polyadenylation usually occurred at A residues, about 100 bases upstream of a trans-splicing acceptor signal. The wild-type polyadenylation site has a cryptic trans-splicing signal about 100 bp downstream: deletion or inversion of this signal results in polyadenylation at multiple sites, upstream of other cryptic trans-splicing signals. The PARP mRNA precursor appears to contain a hierarchy of possible processing signals, the function of cryptic ones being revealed only when the dominant ones are deleted or moved. Correct polyadenylation can be restored by addition of trans-splicing signals from other loci. The results indicate that polyadenylation is coupled to downstream trans splicing but that the products of the trans-splicing reaction are not necessarily functional mRNAs.
Insights
Trypanosome mRNA processing involves trans splicing and polyadenylation. This study shows polyadenylation is linked to trans splicing, but not all trans-spliced products form functional mRNAs.
Area of Science:
- Molecular Biology
- Parasitology
- Gene Expression
Background:
- Trypanosome mRNAs are derived from polycistronic precursors.
- Mature mRNAs are generated through trans splicing and polyadenylation.
Purpose of the Study:
- Investigate the polyadenylation mechanism of procyclic acidic repetitive protein (PARP) mRNA.
- Determine the relationship between polyadenylation and trans splicing in trypanosomes.
Main Methods:
- Transient transfection of constructs containing a chloramphenicol acetyltransferase gene linked to the PARP intergenic region.
- Analysis of polyadenylation sites and trans-splicing signals through genetic manipulation (deletion/inversion).
Main Results:
- Polyadenylation typically occurs upstream of a trans-splicing acceptor signal.
- Deletion or inversion of a downstream cryptic trans-splicing signal leads to alternative polyadenylation sites.
- PARP mRNA precursors exhibit a hierarchy of processing signals.
Conclusions:
- Polyadenylation is coupled to downstream trans splicing in trypanosomes.
- The functional outcome of trans-splicing is not always a mature mRNA.