Characterization of the Crithidia fasciculata mRNA Cycling Sequence Binding Proteins

Abstract

The Crithidia fasciculata cycling sequence binding protein (CSBP) binds with high specificity to sequence elements in several mRNAs that accumulate periodically during the cell cycle. Mutations in these sequence elements abolish both cycling of the mRNA and binding of CSBP. Two genes, CSBPA and CSBPB, encoding putative subunits of CSBP have been cloned and were found to be present in tandem on the same DNA molecule and to be closely related. CSBPA and CSBPB are predicted to encode proteins with sizes of 35.6 and 42.0 kDa, respectively. Both CSBPA and CSBPB proteins have a predicted coiled-coil domain near the N terminus and a novel histidine and cysteine motif near the C terminus. The latter motif is conserved in other trypanosomatid species. Gel sieving chromatography and glycerol gradient sedimentation results indicate that CSBP has a molecular mass in excess of 200 kDa and an extended structure. Recombinant CSBPA and CSBPB also bind specifically to the cycling sequence and together can be reconstituted to give an RNA gel shift similar to that of purified CSBP. Proteins in cell extracts bind to an RNA probe containing six copies of the cycling sequence. The RNA-protein complexes contain both CSBPA and CSBPB, and the binding activity cycles in near synchrony with target mRNA levels. CSBPA and CSBPB mRNA and protein levels show little variation throughout the cell cycle, suggesting that additional factors are involved in the cyclic binding to the cycling sequence elements.Gene organization and expression in trypanosomes differ significantly from those of higher eukaryotes. Trypanosome genes typically lack introns, although a single example of an intron-containing gene in Trypanosoma brucei and Trypanosoma cruzi has been reported recently (15). Also, proteincoding genes are generally organized into long polycistronic transcription units. Polycistronic transcripts are not observed, however, since cleavage, trans-splicing of a 39-nucleotide (nt) spliced leader sequence to the 5Ј end of all mRNAs, and polyadenylation of 3Ј ends occur rapidly to produce individual mRNAs. Consequently, most regulation of mRNA levels is exerted posttranscriptionally (23).The most extensive studies of polycistronic transcription units are those of telomeric expression sites used by the variant surface glycoprotein (VSG) genes during the bloodstream phase of T. brucei. Expression site-associated gene (ESAG) mRNA and VSG mRNA levels differ by more than 100-fold, despite their being transcribed at the same level (13). For parasites such as T. brucei, which undergo morphological and physiological transformations during differentiation of mammalian bloodstream forms to that characteristic of the midgut of the insect vector, many genes show a high level of stagespecific expression. In the case of VSG mRNA, sequences within the 3Ј untranslated region (UTR) of the mRNA were shown to influence gene expression (2, 12). Expression of the T. brucei procyclins, which are the major surface glycoproteins of the insect form of the parasite, was also sh...

Ubiquitination of mRNA cycling sequence binding protein from Leishmania donovani (LdCSBP) modulates the RNA endonuclease activity of its Smr domain

“…The specific detection of the retarded ubiquitinated 6 His-LdCSBP only by anti-ubiquitin antibody (Fig. 2B, a-Ub, lane 5) in the same reaction, further confirmed the covalent modification of LdCSBP through ubiquitination.…”

“…2B, after 2 h of incubation with the S-100 extract and ubiquitin, the mobility of Myc-LdCSBP band was retarded by a distance corresponding to approximately 8 kDa -equivalent to the size of one ubiquitin residue (a-Myc, lanes 1-3), suggesting covalent addition of one molecule of ubiquitin to the protein. The characteristic retardation of LdCSBP band due to ubiquitination was also detected when the reaction was carried out with the 6 His-LdCSBP expressed in Sf9 cells (Fig. 2B, a-His, lanes 6 and 7).…”

“…3A, with increasing time periods, 6 His-LdCSBP-Smr (expressed in Sf9 cells) in reactions containing supercoiled plasmid resulted in increasing amount of linear and open circular forms of the plasmid (lanes 2-4) confirming the nicking DNA endonuclease activity of the Smr domain. Similar activity was also observed with bacterially expressed 6 His-LdCSBP-Smr (Supplementary Fig. S3).…”

“…S3). It was striking to note that the full-length 6 His-LdCSBP did not show any DNA endonuclease activity (Fig. 3B, lanes 2-4) indicating a different behavior of the Smr domain when it is a part of a large protein.…”

“…On the other hand, since LdCSBP is an RNA binding protein, it is an interesting question whether the Smr domain of LdCSBP possesses RNA endonuclease activity. In order to address this question, riboendonuclease activity of 6 His-LdCSBP-Smr (expressed in Sf9 cells) was tested first with a circular RNA of 20 nucleotides as substrate and as shown in Fig. 3C, increasing amounts of the specifically cleaved products of 5 and 3 nucleotides in length were obtained with longer , lanes 1-3).…”