The 3′-terminal region of the mRNAs for VSG and procyclin can confer stage specificity to gene expression in Trypanosoma brucei.

Two Trypanosoma brucei cyclin genes, CYC2 and CYC3, have been isolated by rescue of the Saccharomyces cerevisiae mutant DL1, which is deficient in CLN G 1 cyclin function. CYC2 encodes a 24-kDa protein that has sequence identity to the Neurospora crassa PREG1 and the S. cerevisiae PHO80 cyclin. CYC3 has the most sequence identity to mitotic B-type cyclins from a variety of organisms. Both CYC2 and CYC3 are single-copy genes and expressed in all life cycle stages of the parasite. To determine if CYC2 is found in a complex with previously identified trypanosome cdc2-related kinases (CRKs), the CYC2 gene was fused to the TY epitope tag, integrated into the trypanosome genome, and expressed under inducible control. CYC2ty was found to associate with an active trypanosome CRK complex since CYC2ty bound to leishmanial p12 cks1 , and histone H1 kinase activity was detected in CYC2ty immune-precipitated fractions. Gene knockout experiments provide evidence that CYC2 is an essential gene, and co-immune precipitations together with a two-hybrid interaction assay demonstrated that CYC2 interacts with CRK3. The CRK3⅐CYC2ty complex, the first cyclin-dependent kinase complex identified in trypanosomes, was localized by immune fluorescence to the cytoplasm throughout the cell cycle.

Section: Discussionmentioning

confidence: 99%

Isolation of Trypanosoma brucei CYC2 andCYC3 Cyclin Genes by Rescue of a Yeast G1Cyclin Mutant

Hellemond¹,

Neuville²,

Schwarz³

et al. 2000

“…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), amastin (10), and H2A histone (11) genes in Trypanosoma cruzi. Trans-acting factor, a developmentally regulated U-rich RNA-binding protein involved in selective mRNA destabilization, has recently been identified in T. cruzi (12).…”

mentioning

confidence: 99%

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

Mittra

Sinha

Hines

2003

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

“…However, Northern blots showed that the GP63 mRNAs accumulate to a detectable steady-state level only in bloodstream trypanosomes, as do VSG mRNAs. In the case of VSG mRNAs, their semiconserved 3Ј-UTRs are important in conferring the bloodstream stage specificity to the VSG mRNAs (64). A multiple alignment of the three 3Ј-UTRs of the sequenced trypanosome GP63 genes revealed a high level of homology in those regions.…”

Section: Discussionmentioning

confidence: 99%

“…Our attempts to identify substantive sequence similarities in the 3Ј-UTR sequences of several expressed VSG genes and these three GP63 3Ј-UTRs were not particularly revealing; however, a sequence resemblance to a 14-mer element conserved in all VSG mRNAs was observed immediately upstream of the polyadenylation site of the GP63 genes. The presence of this 14-mer element has been shown to be necessary, but not sufficient, for VSG gene regulation (64).…”

Section: Discussionmentioning

confidence: 99%

African Trypanosomes Have Differentially Expressed Genes Encoding Homologues of the Leishmania GP63 Surface Protease

El-Sayed

Donelson

1997

The genomes of various Leishmania parasites contain tandemly arrayed genes encoding an abundant 63-kDa surface glycoprotein called GP63. Leishmania GP63s are metalloproteases that play an important role in the invasion and survival of the parasites within the macrophage, and their presence on the Leishmania surface has been correlated with resistance to complement-mediated lysis. Here we report the identification of GP63-like genes in African trypanosomes. The predicted trypanosome and Leishmania GP63s share a metalloprotease catalytic site motif of HEXXH as well as 19 cysteines and 10 prolines, implying a conservation of enzymatic activity and secondary/tertiary structure. The trypanosome GP63 genes are transcribed equally in procyclic and bloodstream trypanosomes, but their mRNAs accumulate to a 50-fold higher steady state level in bloodstream trypanosomes, where the ratio of mRNAs for GP63 and variant surface glycoprotein is about 1:150. Transcription of the GP63 genes is sensitive to ␣-amanitin, indicating that they are transcribed by a different polymerase than the variant surface glycoprotein genes. These results lead to a reconsideration of the potential functions of GP63, inasmuch as African trypanosomes are not known to interact with macrophages and do not have an intracellular stage during their life cycle.