Differential response to RNA trans-splicing signals within the phosphoglycerate kinase gene cluster in Trypanosoma brucei

Nikitas Kapotas, Vivian Bellofatto

Research output: Contribution to journalArticlepeer-review

35 Scopus citations

Abstract

In trypanosomatlds, nuclear pre-mRNA splicing is exclusively a trans-spliclng reaction in which a capped, 39 nt exon, the mini-exon, is positioned 5' to an open reading frame. Differential RNA splicing might reflect specific mini-exon and 3' splice site Interactions. To test this hypothesis, we compared the efficiency of mini-exon addition to three natural 3' splice acceptor sites (SASs) located within a single pre-mRNA transcript. In Trypanosoma brucei, the phosphoglycerate kinase A, B and C genes (PGK A, B and C) are co-expressed as three consecutive sequences on a polycistronic pre-mRNA. This pre-mRNA gives rise to unequal amounts of PGK A, B and C mRNAs. When the SAS from each gene was placed upstream of the luciferase open reading frame and the resultant constructs transiently transfected Into T. brucei procyclic cells, luciferase activity levels indicated differential SAS utilization. Enzyme activity was low when the SAS from the A gene was present. Levels were indistinguishable when the B and C SASs were compared. After replacing luciferase with chloramphenicol acetyl transferase in the test constructs, enzyme activities were shown to directly correlate with mRNA amounts. Thus, poor splicing efficiency accounts for the differential expression of the PGK A mRNA during PGK pre-mRNA maturation. This reaction appears to reflect the polypyrimidine pattern within the 3' splice acceptor site.

Original languageEnglish (US)
Pages (from-to)4067-4072
Number of pages6
JournalNucleic acids research
Volume21
Issue number17
DOIs
StatePublished - Aug 25 1993
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Genetics

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