Abstract
A consensus sequence present in the 5'- or 3'-untranslated regions of several Crithidia fasciculata messenger RNAs encoding proteins involved in DNA metabolism has been shown to be necessary for the periodic accumulation of these mRNAs during the cell cycle. A protein complex termed cycling sequence-binding protein (CSBP) has two subunits, CSBPA and CSBPB, and binds the consensus sequence with high specificity. The binding activity of CSBP was shown to vary during the cell cycle in parallel with the levels of putative target mRNAs. Although disruption of the CSBPA gene resulted in loss of both CSBPA and CSBPB, the putative target message levels still continued to vary during the cell cycle. The presence of an additional and distinct binding activity was revealed in these CSBPA null mutant cells. This activity, termed CSBP II, was also expressed in wild-type Crithidia cells. CSBP II has higher binding specificity for the cycling sequence element than the earlier described CSBP complex. Three polypeptides associated with purified CSBP II show specific binding to the cycling sequence. These proteins may represent a family of sequence-specific RNA-binding proteins involved in post-transcriptional regulation.
Highlights
Trypanosomes contain a novel mitochondrial DNA network termed kinetoplast DNA consisting of thousands of minicircle DNA molecules and a small number of maxicircle DNA molecules interlocked to form a huge catenated structure [1]
Western blot analyses were performed with cell lysates from wild-type and mutant cells to confirm the absence of CSBPA protein in the mutant cells (Fig. 1B)
Levels of both CSBPA and CSBPB were decreased in the heterozygote as compared with the level detected in wild-type cells
Summary
Trypanosomes contain a novel mitochondrial DNA network termed kinetoplast DNA consisting of thousands of minicircle DNA molecules and a small number of maxicircle DNA molecules interlocked to form a huge catenated structure [1]. The transcript levels of the genes encoding the large and middle subunits of the nuclear protein RPA (RPA1 and RPA2, a homolog of the human replication protein A), dihydrofolate reductase-thymidylate synthase (DHFR-TS), the kinetoplastspecific topoisomerase II (TOP2), and the histone-like kinetoplast-associated protein 3 have been shown to cycle in parallel, reaching maximum levels during the late G1 and S phases and declining rapidly during the G2 and M phases [15] Transcripts of these genes were found to possess one or more copies of a consensus octameric sequence (C/A)AUAGAA(G/A) with a highly conserved hexameric core in either their 5Ј- or 3Ј-UTR [16, 17]. The putative target mRNAs are found to be most stable at times when the cycling sequence binding activity is high, and the message levels decline sharply in parallel with the decrease in binding activity Based on these observations, we had proposed that the binding of the CSBP protein complex to the octamer sequence might confer a cell cycle-dependent stabilization of transcripts containing these sequence elements. We report here the identification, purification, and biochemical characterization of this CSBP II protein complex
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