Presence of Multiple mRNA Cycling Sequence Element-binding Proteins in Crithidia fasciculata

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.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). Kinetoplast DNA replication occurs through a unique process in which the minicircles are released from the network for replication, and the newly replicated minicircles still containing nicks and gaps are reattached to the network periphery (for recent reviews see Refs. 2 and 3). However, the maxicircles replicate while remaining attached to the network. Unlike in higher eukaryotes where mitochondrial DNA replication occurs throughout the cell cycle, kinetoplast DNA replication in trypanosomes occurs in apparent synchrony with nuclear DNA replication (4). Studies have been undertaken to understand the possible role that cell cycle-dependent coordinated expression of DNA replication genes may play in coordinating nuclear and kinetoplast DNA replication.Regulation of gene expression in trypanosomatids is predominantly post-transcriptional (reviewed in Ref. 5). Polycistronic messages are generated through constitutive transcription of protein-coding genes by RNA polymerase II, which then undergo 5Ј-trans-splicing and 3Ј-polyadenylation to produce the mature mRNA. Multiple points of regulation controlling expression of specific transcripts have been investigated. Cisacting factors that affect mRNA stability have been identified within the 3Ј-untranslated region (UTR) 1 of specific transcripts. AU-rich cis-regulatory elements present in the 3Ј-UTR have been shown to regulate the stability of transcripts of procyclic acidic repetitive proteins (EP and GPEET) or procyclins (6, 7) and the variable surface glycoprotein gene transcripts in Trypanosoma brucei (2, 8) and those of mucin (9

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

“…CSBP II was purified on an RNA affinity column prepared by binding RNA containing six copies of the wild-type cycling sequence CAUA GAAG followed by an (A) 25 tail to oligo(dT) cellulose. Purified proteins were resolved by SDS-10% PAGE as previously described (27). Polypeptide bands of interest were excised from the gel and subjected to in-gel trypsin digestion.…”

“…Gel shift assays were performed with 32 P-labeled RNA probes containing six copies of either the wild-type cycling sequence (CAUAGAAG) or a mutated sequence (CAUAGcAG, where a point mutation is indicated by the lowercase letter). RNA probes were prepared from NotI-linearized plasmids pRM16 and pRM23, containing six copies of either the wild-type cycling sequence (RM16) or its mutated version where the adenine at the sixth position of the octamer has been changed to cytosine (RM23), by using the Maxiscript kit (Ambion) as described elsewhere (27). Binding reactions (20 l) (20) were performed for 20 min at 28°C in the presence of 10 mg of heparin and 10 U of the RNase inhibitor RNasin.…”

“…The binding activity of these proteins varies during the cell cycle in parallel with the levels of putative target mRNAs. Target messages were found to accumulate when the binding activity was high (19,27), suggesting that the variation in the levels of the cycling messages may be a consequence of the cell cycle-dependent periodic binding of the cycling sequence binding proteins to the cycling sequence.…”

“…Closer scrutiny revealed that the 52-kDa band is actually a doublet of closely migrating bands approximately 52 and 55 kDa in size. UV cross-linking of purified CSBP II showed three polypeptides, similar in size to the 68-, 52-and 55-, and 35-kDa proteins, that bind specifically to the wild-type RNA probe (27).…”

“…Two cycling sequence binding activities, cycling sequence binding proteins (CSBP) (19) and CSBP II (27) identified in Crithidia whole-cell extracts, were reported previously. Two subunits of CSBP have been identified, a 37-kDa CSBPA and a 48-kDa CSBPB.…”