The monogenetic kinetoplastid protozoan,<i>Crithidia fasciculate</i>, contains a transcriptionally active, multicopy mini-exon sequence

Muhich, Michael L.; Hughes, Dallas E.; Simpson, Agda M.; Simpson, Larry

doi:10.1093/nar/15.7.3141

Cited by 64 publications

(24 citation statements)

References 36 publications

(32 reference statements)

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“…The gene encoding UMSBP is nuclear, as trans-splicing of miniexon sequences is an attribute of trypanosomatid nuclear transcripts (29). Furthermore, using the UMSBP cDNA as a probe in Southern blot hybridization analysis, we detected a signal with genomic DNA but not with kDNA preparations.…”

Section: Discussionmentioning

confidence: 81%

A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.

Abeliovich¹,

Tzfati²,

Shlomai³

1993

Mol. Cell. Biol.

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Replication of the kinetoplast DNA minicircle light strand initiates at a highly conserved 12-nucleotide sequence, termed the universal minicircle sequence. A Crithidia fasciculata single-stranded DNA-binding protein interacts specifically with the guanine-rich heavy strand of this origin-associated sequence (Y. Tzfati, H. Abeliovich, I. Kapeller, and J. Shlomai, Proc. Natl. Acad. Sci. USA 89:6891-6895, 1992). Using the universal minicircle sequence heavy-strand probe to screen a C. fasciculata cDNA expression library, we have isolated two overlapping cDNA clones encoding the trypanosomatid universal minicircle sequence-binding protein. The complete cDNA sequence defines an open reading frame encoding a 116-amino-acid polypeptide chain consisting of five repetitions of a CCHC zinc finger motif. A significant similarity is found between this universal minicircle sequence-binding protein and two other single-stranded DNA-binding proteins identified in humans and in Leishmania major. All three proteins bind specifically to single-stranded guanine-rich DNA ligands. Partial amino acid sequence of the endogenous protein, purified to homogeneity from C. fasciculata, was identical to that deduced from the cDNA nucleotide sequence. DNA-binding characteristics of the cDNA-encoded fusion protein expressed in bacteria were identical to those of the endogenous C. fasciculata protein. Hybridization analyses reveal that the gene encoding the minicircle origin-binding protein is nuclear and may occur in the C. fasciculata chromosome as a cluster of several structural genes.

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Section: Discussionmentioning

confidence: 81%

A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.

Abeliovich¹,

Tzfati²,

Shlomai³

1993

Mol. Cell. Biol.

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“…The sequence information from the p54 peptides was used to synthesise degenerate antisense primers that were used in different combinations in PCRs against a spliced leader primer (SL: 5′-CGCTATAAATAAGTATCAGTTTCTGTACTTTATTG-3′) that is directed against a common sequence at the 5′ end of all Crithidia mRNAs (Muhich et al, 1987). The following combination gave a correct PCR product: In the first PCR round the primer SW 46 (5′-ACYTCIGGIACIGGRAANACRTC-3′ with R=A/G, Y=T/C, N=A/G/T/C, based on peptide DVFPVPEV) was used against SL and the product was reamplified using the nested Primer SW 42 (5′-TCYTTIGGIGGIATNGTYTCNGA-3′ based on peptide SETIPPKD).…”

Section: Cloning Of P54mentioning

confidence: 99%

Identification of CfNek, a novel member of the NIMA family of cell cycle regulators, as a polypeptide copurifying with tubulin polyglutamylation activity inCrithidia

Westermann

Weber

2002

Journal of Cell Science

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Post-translational glutamylation of tubulin plays an important role in regulating the interaction between microtubules and associated proteins, but so far the enzymes involved in this process have not been cloned from any cellular source. Using a modified purification scheme that employs a hydroxyapaptite chromatography as the final step we identified a 54 kDa band as the major polypeptide copurifying with tubulin polyglutamylation activity from the trypanosomatid Crithidia fasciculata. Based on peptide sequence information we have cloned the corresponding cDNA and identify Crithidia p54 as a novel member (termed CfNek) of the NIMA family of putative cell cycle regulators. CfNek is a protein of 479 amino acids that contains an unusual protein kinase domain that lacks the glycine-rich loop in subdomain I. The protein also harbours a PEST sequence and a pleckstrin homology domain. The tubulin polyglutamylase preparation displays theβ-casein phosphorylation activity typical for NIMA related kinases. Recombinant His-tagged CfNek expressed in Crithidia localises to the flagellar attachment zone/basal body of the parasite. After purification on a Ni2+-column the recombinant enzyme preparation displays ATP-dependent tubulin polyglutamylation activity as well as casein-phosphorylation activity.

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“…Very recently a 5' splice site-U6 RNA interaction was demonstrated to be important for 5' splice site selection in the yeast Saccharomyces [36]; L. collosoma, T. brucei [32]; L. seymouri [5]; T. cruzi, T. vivax [16]; T. rangeli [4]; Leishmania enriettii [33]; Leishmania donovani [50]; Leishmania me-xicana [2]; Herpetomonas samuelpessoai [3]; T. congolense [12]; T. simiae, Leishmania amazonensis, Leishmania major, Leishmania brasiliensis, Leishmania tarentolae [9]; and Endotrypanum schaudinnii LVS9 [20] (27,28). cerevisiae (27,28).…”

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confidence: 99%

trans-spliceosomal U6 RNAs of Crithidia fasciculata and Leptomonas seymouri: deviation from the conserved ACAGAG sequence and potential base pairing with spliced leader RNA.

Wieland

Bindereif

1994

Mol. Cell. Biol.

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U6 RNA genes from the trypanosomatids Crithidia fasciculata and Leptomonas seymouri have been isolated and sequenced. As in Trypanosoma brucei, the U6 RNA genes in both C.fasciculata and L. seymouri are arranged in close linkage with upstream tRNA genes. The U6 RNA sequences from C. fasciculata and L. seymouri deviate in five and three positions, respectively, from the published T. brucei sequence. Interestingly, both C.fasciculata U6 RNA genes carry a C->T change at the second position of the ACAGAG hexanucleotide sequence, which is important for splicing function and has been considered phylogenetically invariable. A compensatory base change of the C. fasciculata spliced leader RNA at the highly conserved 5' splice site position +5, G->A, suggests that an interaction between the 5' splice site region and U6 RNA recently proposed for the yeast cis-splicing system may also occur in trans splicing.Expression of protein-coding genes in trypanosomes involves mRNA processing of a primary transcript, which is usually derived from a long polycistronic transcription unit (for a review, see references 1 and 11). The mature 5' end of each mRNA is generated through trans splicing and the 3' end is generated through polyadenylation. As far as we know, protein-coding genes in trypanosomes are not interrupted by internal introns. Thus there appears to be no conventional cis splicing. Through trans splicing, the 39-nucleotide miniexon sequence at the 5' end of the approximately 140-nucleotide spliced-leader (SL) RNA is joined with each protein-coding exon. trans splicing proceeds through a two-step mechanism of cleavage-ligation reactions, formally analogous to the wellcharacterized cis splicing.A unique component of the trans-splicing machinery is the SL RNA, which occurs in the form of a ribonucleoprotein (RNP) in the cell (13, 31); the core SL RNP is composed of five common proteins, which are also part of the U2 and U4/U6 small nuclear RNPs (snRNPs) (37, 38). The SL RNP has been proposed to play a dual role, functioning as a trans-splicingspecific, possibly Ul-like snRNP and at the same time carrying the miniexon sequence with the 5' splice site (8). How the two trans-splicing substrates are held together within a putative trans spliceosome is unclear; we also know very little about which interactions juxtapose the two splice sites during trans splicing. In the nematode system, there is evidence that an SL-U6 base-pairing interaction is important for trans splicing (25), but it is not clear whether this interaction is conserved in the trypanosome system. U6 RNA undergoes several conformational transitions during the spliceosome cycle. First, it is present in a singular form (39); second, in the U4/U6 snRNP it is stably base paired with U4 RNA (7); third, after the U4-U6 interaction has been disrupted in the spliceosome, U6 interacts by base pairing with U2, which is considered the active conformation of U6 (30). Many studies of the yeast and mammalian cis-splicing systems have provided evidence that the U6 RNA is one of t...

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The monogenetic kinetoplastid protozoan,Crithidia fasciculate, contains a transcriptionally active, multicopy mini-exon sequence

Cited by 64 publications

References 36 publications

A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.

A trypanosomal CCHC-type zinc finger protein which binds the conserved universal sequence of kinetoplast DNA minicircles: isolation and analysis of the complete cDNA from Crithidia fasciculata.

Identification of CfNek, a novel member of the NIMA family of cell cycle regulators, as a polypeptide copurifying with tubulin polyglutamylation activity inCrithidia

trans-spliceosomal U6 RNAs of Crithidia fasciculata and Leptomonas seymouri: deviation from the conserved ACAGAG sequence and potential base pairing with spliced leader RNA.

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