Regulated expression of glycosomal phosphoglycerate kinase in Trypanosoma brucei.
In Trypanosoma brucei, the PGKB and PGKC genes-encoding phosphoglycerate kinase are co-transcribed as part of a polycistronic RNA. PGKB mRNA and the cytosolic PGKB protein are much more abundant in the procyclic life-cycle stage than in bloodstream forms, whereas PGKC mRNA and glycosomal PGKC protein are specific to bloodstream forms. We here show that a sequence between nucleotides 558 and 779 in the 3'-untranslated region of the PGKC mRNA causes low expression of the chloramphenicol acetyltransferase (CAT) reporter gene in procyclic trypanosomes. In procyclics, depletion of the RRP45 component of the exosome (3'-->5' exonuclease complex) or the 5'-->3' exonuclease XRNA increased the abundance of CAT-PGKC mRNA as a consequence of effects on the degradation of precursor and/or mature mRNAs. In bloodstream forms, inhibition of both trans splicing and transcription resulted in immediate exponential decay of PGKC mRNA with a half-life of 46 min. Inhibition of transcription alone gave non-exponential kinetics and inhibition of splicing alone resulted in a longer apparent half-life. We also found that production of mRNAs using T7 polymerase can affect the apparent half-life, and that large amounts of CAT enzyme may be toxic in trypanosomes.
Colasante
Claudia
C
Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany.
Robles
Ana
A
Li
Chi-Ho
CH
Schwede
Angela
A
Benz
Corinna
C
Voncken
Frank
F
Guilbride
D Lys
DL
Clayton
Christine
C
D020413:3' Untranslated Regions; D000818:Animals; D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D015536:Down-Regulation; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D008018:Life Cycle Stages; D008830:Microbodies / Q000201:enzymology*; D008969:Molecular Sequence Data; D010735:Phosphoglycerate Kinase / Q000235:genetics*; D022821:RNA Splice Sites; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016053:RNA, Protozoan / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000254:growth & development
D020413:3' Untranslated Regions; D022821:RNA Splice Sites; D012333:RNA, Messenger; D016053:RNA, Protozoan; D015500:Chloramphenicol O-Acetyltransferase; D010735:Phosphoglycerate Kinase