Characterization of a GTP-binding protein in the ADP-ribosylation factor subfamily from Leishmania tarentolae

Sturm, Nancy R.; Valkenburgh, Hillary Van; Kahn, Richard; Campbell, David A.

doi:10.1016/s0167-4781(98)00150-x

Cited by 8 publications

(8 citation statements)

References 33 publications

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“…Reverse Transcriptase-PCR trans-Splicing Assay-To assay for trans-splicing of mutant-SL RNAs, complementary oligonucleotides were hybridized to Arl mRNA (29) and extended by Moloney murine leukemia virus-reverse transcriptase (RT) to produce templates for PCR analysis (RT-PCR (30)) with a second, SL-specific oligonucleotide. The following oligonucleotides were used: Arl(Ϫ)68, 5Ј-TGCGGATCGC-CTTCTGGCCACC; LtSL5ЈRI, 5Ј-GGGAATTCGCTTTCAACTAACGC-TAT; 30/39 -5ЈHI, 5Ј-GGGATCCTGTATCAGTTTCAGCCT.…”

Section: Methodsmentioning

confidence: 99%

“…B, low levels of trans-splicing detected by RT-PCR. RT-PCR assays were performed on the mutant RNA populations with either nonspecific SL RNA primer (top and middle) or 28/32 tag-specific SL RNA primer (bottom) using the L. tarentolae Arl mRNA (29) as the query template. Control reactions included RNA from a mutant containing tSL coupled with an inactive promoter, Ϫ67/Ϫ58 ϩ tSL (11), -RT, and -RNA reactions.…”

Section: Fig 2 Sl Rna Intron Mutations Affect Trans-splicingmentioning

confidence: 99%

“…In addition, trans-splicing was assayed by RT-PCR (11,29) to detect low levels of splicing (Fig. 2B).…”

Section: Fig 2 Sl Rna Intron Mutations Affect Trans-splicingmentioning

confidence: 99%

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The Role of Intron Structures in trans-Splicing and Cap 4 Formation for the LeishmaniaSpliced Leader RNA

Sturm

Campbell

1999

Journal of Biological Chemistry

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A 39-nucleotide leader is trans-spliced onto all trypanosome nuclear mRNAs. The precursor spliced leader RNA was tested for trans-splicing function in vivo by mutating the intron. We report that in Leishmania tarentolae spliced leader RNA 5 modification is influenced by the primary sequence of stem-loop II, the Smbinding site, and the secondary structure of stem-loop III. The sequence of stem-loop II was found to be important for cap 4 formation and splicing. As in Ascaris, mutagenesis of the bulge nucleotide in stem-loop II was detrimental to trans-splicing. Because restoration of the L. tarentolae stem-loop II structure was not sufficient to restore splicing, this result contrasts the findings in the kinetoplastid Leptomonas, where mutations that restored stem-loop II structure supported splicing. Methylation of the cap 4 structure and splicing was also dependent on both the Sm-binding site and the structure of stem-loop III and was inhibited by incomplete 3 end processing. The critical nature of the L. tarentolae Smbinding site is consistent with its essential role in the Ascaris spliced leader RNA, whereas in Leptomonas mutation of the Sm-binding site and deletion of stem-loop III did not affect trans-splicing. A pathway for Leishmania spliced leader RNA processing and maturation is proposed.Kinetoplastid nuclear gene expression is dependent on the trans-splicing process. The common substrate for all transsplicing reactions is the spliced leader (SL) 1 RNA, also known as the mini-exon derived RNA, whose first 39 nt constitute the 5Ј ends of both mono-and polycistronically synthesized mRNAs (1). The polycistronic pre-mRNAs require trans-splicing to acquire the specialized "cap 4" structure on the SL RNA. The cap 4 consists of a 7 mG attached to the first nucleotide (2), in addition to methylation of the first four and sixth nucleotides of the SL RNA (3-5). These modifications are made to the primary SL RNA and spliced onto the mRNA as part of the 39-nt exon. The cap 4 may have roles in mRNA trans-splicing, transport, stability and translation.The SL RNA contains two functional domains as follows: the exon and the intron or snRNA-like domain (6). The exon sequence is conserved among 38 different members of the order Kinetoplastida (7). Positions 1-9 and 20 -39 of the exon are nearly identical, whereas positions 10 -19 are relatively heterogeneous and characteristically A/T-rich. This conservation cannot be ascribed to internal promoter location in Leishmania (8, 9), as found in Ascaris (10 (7); however, the secondary structure is consistent (16). This structure has been confirmed by physical-chemical and enzymatic studies (17, 18) and examined by mutagenesis (12). An equivalent structure is also conserved in the nematode SL RNAs (16,19). The intron contains a putative Sm-binding site (16), an element found in the small nuclear RNAs of higher eukaryotes but apparently lacking in all U-RNAs of kinetoplastids (20) except U5 RNA (21, 22). The Sm-binding element is required for SL RNA trans-splicing in Ascaris (23...

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Section: Methodsmentioning

confidence: 99%

Section: Fig 2 Sl Rna Intron Mutations Affect Trans-splicingmentioning

confidence: 99%

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The Role of Intron Structures in trans-Splicing and Cap 4 Formation for the LeishmaniaSpliced Leader RNA

Sturm

Campbell

1999

Journal of Biological Chemistry

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“…Other members of the ARF family appear to perform related cellular functions, although they differ somewhat in membrane binding specificity and associated protein cofactors. ARL proteins have been particularly difficult to analyze from a functional perspective (Hong et al, 1998;Sturm et al, 1998). No specific function has been assigned to ARLs from Drosophila, humans, rat, or mouse.…”

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confidence: 99%

The TITAN5 Gene of Arabidopsis Encodes a Protein Related to the ADP Ribosylation Factor Family of GTP Binding Proteins

et al. 2000

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The titan ( ttn ) mutants of Arabidopsis exhibit dramatic alterations in mitosis and cell cycle control during seed development. Endosperm development in these mutants is characterized by the formation of giant polyploid nuclei with enlarged nucleoli. Embryo development is accompanied by significant cell enlargement in some mutants ( ttn1 and ttn5 ) but not others ( ttn2 and ttn3). We describe here the molecular cloning of TTN5 using a T-DNA-tagged allele. A second allele with a similar phenotype contains a nonsense mutation in the same coding region. The predicted protein is related to ADP ribosylation factors (ARFs), members of the RAS family of small GTP binding proteins that regulate various cellular functions in eukaryotes. TTN5 is most closely related in sequence to the ARL2 class of ARF-like proteins isolated from humans, rats, and mice. Although the cellular functions of ARL proteins remain unclear, the ttn5 phenotype is consistent with the known roles of ARFs in the regulation of intracellular vesicle transport. INTRODUCTIONEmbryo-defective mutants of Arabidopsis have been used for many years to identify genes with essential functions during plant embryogenesis (Goldberg et al., 1994;Jurgens et al., 1994;Meinke, 1995). Recent advances in gene isolation have made it possible to recover increasing numbers of these genes and study their role in plant growth and development (Li and Thomas, 1998;Lotan et al., 1998;Patton et al., 1998;Uwer et al., 1998;Albert et al., 1999). Development of the female gametophyte and early endosperm in Arabidopsis has also been subjected to extensive genetic analysis (Drews et al., 1998;Berger, 1999). Particular attention has been given to mutants with defects in gene imprinting and mutants in which endosperm development begins in the absence of fertilization (reviewed in Preuss, 1999). Molecular cloning of the medea ( med ) and fertilization-independent endosperm and seed ( fie and fis , respectively) genes (Grossniklaus et al., 1998;Kiyosue et al., 1999;Luo et al., 1999;Ohad et al., 1999) has renewed interest in the biochemical signals associated with fertilization and the coordinated development of the gametophyte, embryo, and endosperm tissue in plants.The titan ( ttn ) mutants of Arabidopsis represent another interesting class of mutants with defects in embryo and endosperm development (Liu and Meinke, 1998). The most striking feature of these mutants is the formation of giant endosperm nuclei and nucleoli during early stages of seed development. Embryo development is arrested shortly after fertilization in most ttn mutants and in some cases is accompanied by dramatic cell enlargement. Three nonallelic ttn mutants ( ttn1 , ttn2 , and ttn3 ) with related but distinct phenotypes were first described by Liu and Meinke (1998). The ttn1 phenotype includes extraordinary enlargement of nuclei in the embryo and endosperm, similar enlargement of cells in the arrested embryo, and a disruption of endosperm nuclear migration to the chalazal end of the seed. Defects in ttn2 are limited...

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“…However, this division is still obscure for plants, fungi and protists. Two reports were published describing Arl proteins in leishmanias (Sturm et al 1998;Cuvillier et al 2000). One of them, LdARL-3A, plays an essential role in the biogenesis of the flagellum of Leishmania donovani (Cuvillier et al 2000).…”

Section: Discussionmentioning

confidence: 99%

TcArf1: a Trypanosoma cruzi ADP-ribosylation factor

et al. 2003

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Arfs (ADP-ribosylation factors) are conserved GTP-binding proteins involved in the control of coatomers assembling in budding vesicles in the eukaryotic secretory pathway and during some endocytic events. Here, we describe the gene for an Arf-homologue from the unicellular kinetoplastid parasite Trypanosoma cruzi, named TcArf1. TcArf1 is present in a discrete copy number in the T. cruzi genome and is expressed as a 1-kb transcript in the three main life forms of the parasite. Increased mRNA expression in epimastigotes may correlate with abundant protein biosynthesis and endocytosis in this stage.

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Characterization of a GTP-binding protein in the ADP-ribosylation factor subfamily from Leishmania tarentolae

Cited by 8 publications

References 33 publications

The Role of Intron Structures in trans-Splicing and Cap 4 Formation for the LeishmaniaSpliced Leader RNA

The Role of Intron Structures in trans-Splicing and Cap 4 Formation for the LeishmaniaSpliced Leader RNA

The TITAN5 Gene of Arabidopsis Encodes a Protein Related to the ADP Ribosylation Factor Family of GTP Binding Proteins

TcArf1: a Trypanosoma cruzi ADP-ribosylation factor

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