Presence of a Poly(A) Binding Protein and Two Proteins with Cell Cycle-Dependent Phosphorylation in Crithidia fasciculata mRNA Cycling Sequence Binding Protein II

Abstract

Crithidia fasciculata cycling sequence binding proteins (CSBP) have been shown to bind with high specificity to sequence elements present in several mRNAs that accumulate periodically during the cell cycle. The first described CSBP has subunits of 35.6 (CSBPA) and 42 kDa (CSBPB). A second distinct binding protein termed CSBP II has been purified from CSBPA null mutant cells, lacking both CSBPA and CSBPB proteins, and contains three major polypeptides with predicted molecular masses of 63, 44.5, and 33 kDa. Polypeptides of identical size were radiolabeled in UV cross-linking assays performed with purified CSBP II and 32 P-labeled RNA probes containing six copies of the cycling sequence. The CSBP II binding activity was found to cycle in parallel with target mRNA levels during progression through the cell cycle. We have cloned genes encoding these three CSBP II proteins, termed RBP63, RBP45, and RBP33, and characterized their binding properties. The RBP63 protein is a member of the poly(A) binding protein family. Homologs of RBP45 and RBP33 proteins were found only among the kinetoplastids. Both RBP45 and RBP33 proteins and their homologs have a conserved carboxy-terminal half that contains a PSP1-like domain. All three CSBP II proteins show specificity for binding the wild-type cycling sequence in vitro. RBP45 and RBP33 are phosphoproteins, and RBP45 has been found to bind in vivo specifically to target mRNA containing cycling sequences. The levels of phosphorylation of both RBP45 and RBP33 were found to cycle during the cell cycle.Kinetoplastid parasites are one of the earliest diverging organisms containing a single mitochondrion and consequently have many unique biological features (35). The genomic structure and mechanisms of regulation of gene expression observed in trypanosomes and other kinetoplastids are significantly different from those in other eukaryotes. Although the majority of the protein coding genes are transcribed by RNA polymerase II, well-defined RNA polymerase II promoters in these organisms have so far remained elusive, with the only exception being the spliced leader promoter (13). Analysis of the distribution and orientation of genes in the Leishmania genome has revealed that most genes in these organisms are organized into long clusters on the same DNA strand and are transcribed from putative bidirectional promoters (23,25,29). Constitutive transcription results in the generation of long polycistronic messages that are then processed further to produce mature monocistronic messages by two physically coupled events: 5Ј trans splicing and 3Ј adenylation (16,24,38). The trans-splicing mechanism introduces a spliced leader sequence of 39 nucleotides that includes a 5Ј RNA cap (37).

The Cell Cycle Regulated Transcriptome of Trypanosoma brucei

“…However, predicted RBPs did not feature prominently on the list of regulated transcripts, which argues against a general model of cell cycle-coupled regulatory cascades in which RBPs bind to each other's transcripts and regulate transcript stability (although regulation of translation is one possibility that we did not investigate). Perhaps post-translational modifications such as phosphorylation regulate RBP activity, as has been suggested for the CSBP II complex [15]. Two notable exceptions to this lack of transcript-level regulation of RBPs are PUF9, an RBP that mediates cell-cycle regulation of a small group of downstream targets [16], and which was observed here to be regulated at the transcript level by 1.9-fold; and Tb927.5.760, the homologue of the C. fasciculata RBP45 subunit of CSBPII which is also responsible for cell cycle regulation of cognate transcripts [15], showing upregulation of ∼1.9 fold in early G1 phase.…”

A Mitochondrial DNA Primase Is Essential for Cell Growth and Kinetoplast DNA Replication in Trypanosoma brucei

“…Since all known DNA polymerases are unable to initiate a DNA strand, it is vital to have a kinetoplastid mitochondrial DNA primase in order to allow the development of an in vitro mitochondrial DNA replication system. A search of the T. brucei genome revealed a putative mitochondrial DNA primase gene (PRI1) with multiple copies of a conserved octamer consensus sequence in the 3Ј flanking sequence that has been shown to be required for the posttran- (33,34,36,37,42). The presence of the consensus sequence in flanking sequences in Leishmania major was also shown to correlate with S-phase expression of the mRNA (26,64).…”

“…We initially identified a gene (Tb927.8.2550) in the GeneDB T. brucei genome database that contains two sequence elements (CATAGAGG and CATAG AAG) within approximately 500 nucleotides of the stop codon that are similar to the conserved octamer sequence (C/A)AU AGAA(G/A) which has been shown to be required for cyclic expression of several S-phase-expressed genes in the related parasite C. fasciculata (26,33,34,36,37). This sequence was also found to be predictive of S-phase gene expression in Leishmania major (26,64).…”

Functional Characterization of Three Leishmania Poly(A) Binding Protein Homologues with Distinct Binding Properties to RNA and Protein Partners

“…recognizes sequence elements in the untranslated regions of mRNAs that accumulate periodically during the cell cycle (46). Again, in T. cruzi, its orthologue was found to interact directly with TcUBP-1, an RNA binding protein that binds to AU-rich sequences in the 3Ј UTRs of mRNAs, which are selectively destabilized in the trypomastigote stage of the parasite (21).…”