Biomedical subjects

V W Pollard

Publications and source records attributed to V W Pollard.

10 recordsLinked to original sources

PubMed2000

Mutational definition of RNA-binding and protein-protein interaction domains of heterogeneous nuclear RNP C1.

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.

Amino Acid Sequence↗

PubMed2000

Trypanosoma brucei guide RNA poly(U) tail formation is stabilized by cognate mRNA.

Guide RNAs (gRNAs) are small RNAs that provide specificity for uridine addition and deletion during mRNA editing in trypanosomes. Terminal uridylyl transferase (TUTase) adds uridines to pre-mRNAs during RNA editing and adds a poly(U) tail to the 3' end of gRNAs. The poly(U) tail may stabilize the association of gRNAs with cognate mRNA during editing. Both TUTase and gRNAs associate with two ribonucleoprotein complexes, I (19S) and II (35S to 40S). Complex II is believed to be the fully assembled active editing complex, since it contains pre-edited mRNA and enzymes thought necessary for editing. Purification of TUTase from mitochondrial extracts resulted in the identification of two chromatographically distinct TUTase activities. Stable single-uridine addition to different substrate RNAs is performed by the 19S complex, despite the presence of a uridine-specific 3' exonuclease within this complex. Multiple uridines are added to substrate RNAs by a 10S particle that may be an unstable subunit of complex I lacking the uridine-specific 3' exonuclease. Multiple uridines could be stably added onto gRNAs by complex I when the cognate mRNA is present. We propose a model in which the purine-rich region of the cognate mRNA protects the uridine tail from a uridine exonuclease activity that is present within the complex. To test this model, we have mutated the purine-rich region of the pre-mRNA to abolish base-pairing interaction with the poly(U) tail of the gRNA. This RNA fails to protect the uridine tail of the gRNA from exoribonucleolytic trimming and is consistent with a role for the purine-rich region of the mRNA in gRNA maturation.

Animals↗

PubMed1999

Rev-mediated nuclear export of RNA is dominant over nuclear retention and is coupled to the Ran-GTPase cycle.

The human immunodeficiency virus type-1 Rev protein induces the nuclear export of intron-containing viral mRNAs that harbor its binding site, the Rev response element (RRE). A leucine-rich region of Rev, the activation domain, is essential for function and has been shown to be a nuclear export signal (NES). Although Rev exports viral RNAs that resemble cellular mRNAs, competition studies performed using microinjected Xenopus laevis oocytes have previously indicated that Rev utilizes a non-mRNA export pathway. Here, we show that Rev is able to induce the export of both spliceable and non-spliceable RRE-containing pre-mRNAs and that this activity is not dependent on the location of the RRE within the RNA. Importantly, even RNA molecules of different classes, such as U3 snoRNA and U6 snRNA, which are retained in the nucleus by non-pre-mRNA mechanisms, are exported to the cytoplasm in response to Rev. Consistent with the notion that Rev-mediated export of RRE-containing RNA is mechanistically distinct from the export of processed cellular mRNA, a chimeric Rev protein in which its NES is replaced by the NES of hnRNP A1 does not induce the export of a Rev-responsive mRNA. Finally, we demonstrate that Rev/RRE-activated RNA export is, like other nuclear export pathways, linked to the Ran-GTPase cycle.

Animals↗

PubMed1998

Kinetoplastid RNA-editing-associated protein 1 (REAP-1): a novel editing complex protein with repetitive domains.

Kinetoplastid RNA editing consists of the addition or deletion of uridines at specific sites within mitochondrial mRNAs. This unusual RNA processing event is catalyzed by a ribonucleoprotein (RNP) complex that includes editing site-specific endoribonuclease, RNA ligase and terminal uridylnucleotidyl transferase (Tutase) among its essential enzymatic activities. To identify the components of this RNP, monoclonal antibodies were raised against partially purified editing complexes. One antibody reacts with a mitochondrially located 45 kDa polypeptide (p45) which contains a conserved repetitive amino acid domain. p45 co-purifies with RNA ligase and Tutase in a large ( approximately 700 kDa) RNP, and anti-p45 antibody inhibits in vitro RNA editing. Thus, p45 is the first kinetoplastid RNA-editing-associated protein (REAP-1) that has been cloned and identified as a protein component of a functional editing complex.

Amino Acid Sequence↗

PubMed1998

The HIV-1 Rev protein.

The nuclear export of intron-containing HIV-1 RNA is critically dependent on the activity of Rev, a virally encoded sequence-specific RNA-binding protein. Rev shuttles between the nucleus and the cytoplasm and harbors both a nuclear localization signal and a nuclear export signal. These essential peptide motifs have now been shown to function by accessing cellular signal-mediated pathways for nuclear import and nuclear export. HIV-1 Rev therefore represents an excellent system with which to study aspects of transport across the nuclear envelope.

Alternative Splicing↗

PubMed1996

A novel receptor-mediated nuclear protein import pathway.

Targeting of most nuclear proteins to the cell nucleus is initiated by interaction between the classical nuclear localization signals (NLSs) contained within them and the importin NLS receptor complex. We have recently delineated a novel 38 amino acid transport signal in the hnRNP A1 protein, termed M9, which confers bidirectional transport across the nuclear envelope. We show here that M9-mediated nuclear import occurs by a novel pathway that is independent of the well-characterized, importin-mediated classical NLS pathway. Additionally, we have identified a specific M9-interacting protein, termed transportin, which binds to wild-type M9 but not to transport-defective M9 mutants. Transportin is a 90 kDa protein, distantly related to importin beta, and we show that it mediates the nuclear import of M9-containing proteins. These findings demonstrate that there are at least two receptor-mediated nuclear protein import pathways. Furthermore, as hnRNP A1 likely participates in mRNA export, it raises the possibility that transportin is a mediator of this process as well.

Amino Acid Sequence↗

PubMed1993

RNA editing in kinetoplastid mitochondria.

RNA editing in the mitochondrion of kinetoplastid protozoa results in the posttranscriptional addition and deletion of uridine residues in mRNAs. Editing of mRNAs can lead to the formation of initiation codons for mitochondrial translation, the correction of frame-shifted genes at the RNA level, and in extensively edited mRNAs, the formation of complete reading frames. Kinetoplastid RNA editing requires that genetic information from two or more separately transcribed genes be brought together to form the mature, edited mRNA. The information necessary for the proper insertion or deletion of uridines in the mRNA is present in small mitochondrial transcripts termed guide RNAs (gRNAs). Editing of mRNAs appears to be associated with a high molecular weight complex, called the editosome, containing specific gRNAs, unedited mRNAs, and proteins. Editing is likely a two-step process involving first the breakage of a phosphodiester bond at the editing site and formation of a chimeric molecule with a gRNA covalently joined to the 5' end of the 3' portion of an mRNA. The chimera is resolved by the rejoining of the 5' end of the mRNA to the 3' portion of the mRNA with the addition or deletion of a uridine at the junction point. Two models are proposed for the biochemical mechanism of RNA editing. The first is an enzymatic cascade of cleavage and ligation while the other supports successive rounds of transesterification. The obvious functional necessity for editing in kinetoplastid mitochondria is the formation of translatable mRNAs. Far less clear is the evolutionary origin of editing and the role editing plays in regulating mitochondrial gene expression.

Animals↗

PubMed1992

Native mRNA editing complexes from Trypanosoma brucei mitochondria.

The aim of this study was to identify multicomponent complexes involved in kinetoplastid mitochondrial mRNA editing. Mitochondrial extracts from Trypanosoma brucei were fractionated on 10-30% glycerol gradients and assayed for RNAs and activities potentially involved in editing, including pre-edited mRNA, guide RNA (gRNA), endonuclease, terminal uridylyltransferase (TUTase), RNA ligase and gRNA-mRNA chimera-forming activities. These experiments suggest that two distinct editing complexes exist. Complex I (19S) consists of gRNA, TUTase, RNA ligase and chimera-forming activity. Complex II (35-40S) is composed of gRNA, preedited mRNA, RNA ligase and chimera-forming activity. These studies provide the first evidence that editing occurs in a multicomponent complex. The possible roles of complex I, complex II and RNA ligase in editing are discussed.

Animals↗

PubMed1991

Trypanosoma equiperdum minicircles encode three distinct primary transcripts which exhibit guide RNA characteristics.

The mitochondrial DNA of trypanosomes is composed of maxicircle and minicircle DNAs catenated into a network, called the kinetoplast. Maxicircles encode proteins and RNAs necessary for mitochondrial assembly. Minicircles encode small transcripts which are believed to serve as guide RNAs in the process of RNA editing of maxicircle transcripts. Trypanosoma equiperdum minicircles contain three transcription units which produce three distinct transcripts. The genes for these transcripts are flanked by imperfect 18-bp repeats separated by approximately 110 bp. The transcripts have a 5' triphosphate, indicating that they are primary transcripts. Minicircle transcription initiates at a purine within a conserved sequence, 5'-AYAYA-3', where Y is a pyrimidine, 32 bp from the upstream inverted repeat, suggesting that the repeats may function in transcript initiation. Transcripts from a single minicircle transcription unit range in size from 55 to 70 nucleotides. This size heterogeneity within a single sequence class is due to the variable length of nontemplated uridine residues composing a 3' tail. The size range and heterogeneous polyuridylate 3' end of the minicircle transcripts appear to be conserved features and may be related to transcript function.

Animals↗

PubMed1990

Organization of minicircle genes for guide RNAs in Trypanosoma brucei.

We have identified four T. brucei minicircle sequences that are complementary to cytochrome oxidase III (COIII) edited mRNA sequence and have shown the existence of transcripts from three of these minicircle sequences. These minicircle transcripts potentially serve as guide RNAs (gRNAs) for RNA editing of the COIII transcript. These gRNAs range in size from 55 to 70 nucleotides, are heterogeneous in sequence, and have a 5' terminal triphosphate. The genes for these gRNAs are flanked by imperfect 18 bp repeats separated by approximately 110 bp. Transcription initiates at the first purine within a conserved sequence, 5'-RYA-YA-3', 31 or 32 bp from the upstream inverted repeat. We propose that these 18 bp inverted repeats are important for minicircle gRNA expression in T. brucei.

Animals↗