scholarly journals Characterization of nuclear polyadenylated RNA-binding proteins in Saccharomyces cerevisiae.

1994 ◽  
Vol 127 (5) ◽  
pp. 1173-1184 ◽  
Author(s):  
S M Wilson ◽  
K V Datar ◽  
M R Paddy ◽  
J R Swedlow ◽  
M S Swanson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Uv Light ◽  
Three Dimensional ◽  
Polyadenylated Rna ◽  
Heterogeneous Nuclear Ribonucleoproteins ◽  
Nuclear Rna Processing

To study the functions of heterogeneous nuclear ribonucleoproteins (hnRNPs), we have characterized nuclear polyadenylated RNA-binding (Nab) proteins from Saccharomyces cerevisiae. Nab1p, Nab2p, and Nab3p were isolated by a method which uses UV light to cross-link proteins directly bound to poly(A)+ RNA in vivo. We have previously characterized Nab2p, and demonstrated that it is structurally related to human hnRNPs. Here we report that Nab1p is identical to the Np13p/Nop3p protein recently implicated in both nucleocytoplasmic protein shuttling and pre-rRNA processing, and characterize a new nuclear polyadenylated RNA-binding protein, Nab3p. The intranuclear distributions of the Nab proteins were analyzed by three-dimensional immunofluorescence optical microscopy. All three Nab proteins are predominantly localized within the nucleoplasm in a pattern similar to the distribution of hnRNPs in human cells. The NAB3 gene is essential for cell viability and encodes an acidic ribonucleoprotein. Loss of Nab3p by growth of a GAL::nab3 mutant strain in glucose results in a decrease in the amount of mature ACT1, CYH2, and TPI1 mRNAs, a concomitant accumulation of unspliced ACT1 pre-mRNA, and an increase in the ratio of unspliced CYH2 pre-mRNA to mRNA. These results suggest that the Nab proteins may be required for packaging pre-mRNAs into ribonucleoprotein structures amenable to efficient nuclear RNA processing.

PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (10) ◽  
pp. 6102-6113
Author(s):  
J T Anderson ◽  
M R Paddy ◽  
M S Swanson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Uv Light ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Rna Binding Protein ◽  
Polyadenylated Rna ◽  
Polyadenylated Rnas

Proteins that directly associate with nuclear polyadenylated RNAs, or heterogeneous nuclear RNA-binding proteins (hnRNPs), and those that associate with cytoplasmic mRNAs, or mRNA-binding proteins (mRNPs), play important roles in regulating gene expression at the posttranscriptional level. Previous work with a variety of eukaryotic cells has demonstrated that hnRNPs are localized predominantly within the nucleus whereas mRNPs are cytoplasmic. While studying proteins associated with polyadenylated RNAs in Saccharomyces cerevisiae, we discovered an abundant polyuridylate-binding protein, PUB1, which appears to be both an hnRNP and an mRNP. PUB1 and PAB1, the polyadenylate tail-binding protein, are the two major proteins cross-linked by UV light to polyadenylated RNAs in vivo. The deduced primary structure of PUB1 indicates that it is a member of the ribonucleoprotein consensus sequence family of RNA-binding proteins and is structurally related to the human hnRNP M proteins. Even though the PUB1 protein is a major cellular polyadenylated RNA-binding protein, it is nonessential for cell growth. Indirect cellular immunofluorescence combined with digital image processing allowed a detailed comparison of the intracellular distributions of PUB1 and PAB1. While PAB1 is predominantly, and relatively uniformly, distributed within the cytoplasm, PUB1 is localized in a nonuniform pattern throughout both the nucleus and the cytoplasm. The cytoplasmic distribution of PUB1 is considerably more discontinuous than that of PAB1. Furthermore, sucrose gradient sedimentation analysis demonstrates that PAB1 cofractionates with polyribosomes whereas PUB1 does not. These results suggest that PUB1 is both an hnRNP and an mRNP and that it may be stably bound to a translationally inactive subpopulation of mRNAs within the cytoplasm.

scholarly journals PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (10) ◽  
pp. 6102-6113 ◽  
Author(s):  
J T Anderson ◽  
M R Paddy ◽  
M S Swanson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Uv Light ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Rna Binding Protein ◽  
Polyadenylated Rna ◽  
Polyadenylated Rnas

Proteins that directly associate with nuclear polyadenylated RNAs, or heterogeneous nuclear RNA-binding proteins (hnRNPs), and those that associate with cytoplasmic mRNAs, or mRNA-binding proteins (mRNPs), play important roles in regulating gene expression at the posttranscriptional level. Previous work with a variety of eukaryotic cells has demonstrated that hnRNPs are localized predominantly within the nucleus whereas mRNPs are cytoplasmic. While studying proteins associated with polyadenylated RNAs in Saccharomyces cerevisiae, we discovered an abundant polyuridylate-binding protein, PUB1, which appears to be both an hnRNP and an mRNP. PUB1 and PAB1, the polyadenylate tail-binding protein, are the two major proteins cross-linked by UV light to polyadenylated RNAs in vivo. The deduced primary structure of PUB1 indicates that it is a member of the ribonucleoprotein consensus sequence family of RNA-binding proteins and is structurally related to the human hnRNP M proteins. Even though the PUB1 protein is a major cellular polyadenylated RNA-binding protein, it is nonessential for cell growth. Indirect cellular immunofluorescence combined with digital image processing allowed a detailed comparison of the intracellular distributions of PUB1 and PAB1. While PAB1 is predominantly, and relatively uniformly, distributed within the cytoplasm, PUB1 is localized in a nonuniform pattern throughout both the nucleus and the cytoplasm. The cytoplasmic distribution of PUB1 is considerably more discontinuous than that of PAB1. Furthermore, sucrose gradient sedimentation analysis demonstrates that PAB1 cofractionates with polyribosomes whereas PUB1 does not. These results suggest that PUB1 is both an hnRNP and an mRNP and that it may be stably bound to a translationally inactive subpopulation of mRNAs within the cytoplasm.

NAB2: a yeast nuclear polyadenylated RNA-binding protein essential for cell viability

1993 ◽  
Vol 13 (5) ◽  
pp. 2730-2741 ◽  
Author(s):  
J T Anderson ◽  
S M Wilson ◽  
K V Datar ◽  
M S Swanson
Keyword(s):  
Rna Binding ◽  
Uv Light ◽  
Polyclonal Antibodies ◽  
Yeast Cells ◽  
Binding Studies ◽  
Single Stranded Dna ◽  
Binding Motifs ◽  
Polyadenylated Rna ◽  
Nuclear Rna

A variety of nuclear ribonucleoproteins are believed to associate directly with nascent RNA polymerase II transcripts and remain associated during subsequent nuclear RNA processing reactions, including pre-mRNA polyadenylation and splicing as well as nucleocytoplasmic mRNA transport. To investigate the functions of these proteins by using a combined biochemical and genetic approach, we have isolated nuclear polyadenylated RNA-binding (NAB) proteins from Saccharomyces cerevisiae. Living yeast cells were irradiated with UV light to covalently cross-link proteins intimately associated with RNA in vivo. Polyadenylated RNAs were then selectively purified, and the covalent RNA-protein complexes were used to elicit antibodies in mice. Both monoclonal and polyclonal antibodies which detect a variety of NAB proteins were prepared. Here we characterize one of these proteins, NAB2. NAB2 is one of the major proteins associated with nuclear polyadenylated RNA in vivo, as detected by UV light-induced cross-linking. Cellular immunofluorescence, using both monoclonal and polyclonal antibodies, demonstrates that the NAB2 protein is localized within the nucleus. The deduced primary structure of NAB2 indicates that it is composed of at least two distinct types of RNA-binding motifs: (i) an RGG box recently described in a variety of heterogeneous nuclear RNA-, pre-rRNA-, mRNA-, and small nucleolar RNA-binding proteins and (ii) CCCH motif repeats related to the zinc-binding motifs of the largest subunit of RNA polymerases I, II, and III. In vitro RNA homopolymer/single-stranded DNA binding studies indicate that although both the RGG box and CCCH motifs bind poly(G), poly(U), and single-stranded DNA, the CCCH motifs also bind to poly(A). NAB2 is located on chromosome VII within a cluster of ribonucleoprotein genes, and its expression is essential for cell growth.

scholarly journals A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability.

1987 ◽  
Vol 7 (9) ◽  
pp. 3268-3276 ◽  
Author(s):  
A B Sachs ◽  
R W Davis ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Association Constant ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
High Affinity ◽  
Terminal Domain ◽  
Necessary And Sufficient

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

scholarly journals A novel methyltransferase (Hmt1p) modifies poly(A)+-RNA-binding proteins.

1996 ◽  
Vol 16 (7) ◽  
pp. 3668-3678 ◽  
Author(s):  
M F Henry ◽  
P A Silver
Keyword(s):  
Normal Cell ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Temperature Sensitive ◽  
Arginine Residues ◽  
Cognate Gene ◽  
Heterogeneous Nuclear Ribonucleoproteins

RNA-binding proteins play many essential roles in the metabolism of nuclear pre-mRNA. As such, they demonstrate a myriad of dynamic behaviors and modifications. In particular, heterogeneous nuclear ribonucleoproteins (hnRNPs) contain the bulk of methylated arginine residues in eukaryotic cells. We have identified the first eukaryotic hnRNP-specific methyltransferase via a genetic screen for proteins that interact with an abundant poly(A)+-RNA-binding protein termed Npl3p. We have previously shown that npl3-1 mutants are temperature sensitive for growth and defective for export of mRNA from the nucleus. New mutants in interacting genes were isolated by their failure to survive in the presence of the npl3-1 allele. Four alleles of the same gene were identified in this manner. Cloning of the cognate gene revealed an encoded protein with similarity to methyltransferases that was termed HMT1 for hnRNP methyltransferase. HMT1 is not required for normal cell viability except when NPL3 is also defective. The Hmt1 protein is located in the nucleus. We demonstrate that Npl3p is methylated by Hmt1p both in vivo and in vitro. These findings now allow further exploration of the function of this previously uncharacterized class of enzymes.

scholarly journals Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins

1987 ◽  
Vol 7 (8) ◽  
pp. 2947-2955
Author(s):  
A Y Jong ◽  
M W Clark ◽  
M Gilbert ◽  
A Oehm ◽  
J L Campbell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Nucleic Acid ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nucleic Acid Binding ◽  
Dna And Rna ◽  
C Terminus

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.

A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability

1987 ◽  
Vol 7 (9) ◽  
pp. 3268-3276 ◽  
Author(s):  
A B Sachs ◽  
R W Davis ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Association Constant ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
High Affinity ◽  
Terminal Domain ◽  
Necessary And Sufficient

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

Differential binding of heterogeneous nuclear ribonucleoproteins to mRNA precursors prior to spliceosome assembly in vitro

1992 ◽  
Vol 12 (7) ◽  
pp. 3165-3175
Author(s):  
M Bennett ◽  
S Piñol-Roma ◽  
D Staknis ◽  
G Dreyfuss ◽  
R Reed
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Packaging ◽  
Splice Site Selection ◽  
Spliceosome Assembly ◽  
Hnrnp Proteins ◽  
Heterogeneous Nuclear Ribonucleoproteins

We have investigated the composition of the earliest detectable complex (H) assembled on pre-mRNA during the in vitro splicing reaction. We show that most of the proteins in this complex correspond to heterogeneous nuclear ribonucleoproteins (hnRNP), a set of abundant RNA-binding proteins that bind nascent RNA polymerase II transcripts in vivo. Thus, these studies establish a direct parallel between the initial events of RNA processing in vitro and in vivo. In contrast to previous studies, in which total hnRNP particles were isolated from mammalian nuclei, we determined the hnRNP composition of complexes assembled on individual RNAs of defined sequence. We found that a unique combination of hnRNP proteins is associated with each RNA. Thus, our data provide direct evidence for transcript-dependent assembly of pre-mRNA in hnRNP complexes. The observation that pre-mRNA is differentially bound by hnRNP proteins prior to spliceosome assembly suggests the possibility that RNA packaging could play a central role in the mechanism of splice site selection, as well as other posttranscriptional events.

scholarly journals Potential RNA Binding Proteins in Saccharomyces cerevisiae Identified as Suppressors of Temperature-Sensitive Mutations in NPL3

Genetics ◽  
1996 ◽  
Vol 142 (1) ◽  
pp. 103-115 ◽  
Author(s):  
Michael Henry ◽  
Christina Z Borland ◽  
Mark Bossie ◽  
Pamela A Silver
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Import ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Heterogeneous Nuclear Ribonucleoproteins ◽  
Rna Export ◽  
Complementation Groups ◽  
Export Protein

The NPL3 gene of the yeast Saccharomyces cerevisiae encodes a protein with similarity to heterogeneous nuclear ribonucleoproteins (hnRNPs). Npl3p has been implicated in many nuclear-related events including RNA export, protein import, and rRNA processing. Several temperature-sensitive alleles of NPL3 have been isolated. We now report the sequence of these alleles. For one allele, npl3-1, four complementation groups of suppressors have been isolated. The cognate genes for the two recessive mutants were cloned. One of these is the previously known RNA15, which, like NPL3, also encodes a protein with similarity to the vertebrate hnRNP A/B protein family. The other suppressor corresponds to a newly defined gene we term HRP1, which also encodes a protein with similarity to the hnRNP A/B proteins of vertebrates. Mutations in HRP1 suppress all npl3 temperature-sensitive alleles but do not bypass an npl3 null allele. We show that HRP1 is essential for cell growth and that the corresponding protein is located in the nucleus. The discovery of two hnRNP homologues that can partially suppress the function of Npl3p, also an RNA binding protein, will be discussed in terms of the possible roles for Npl3p in RNA metabolism.

scholarly journals Differential binding of heterogeneous nuclear ribonucleoproteins to mRNA precursors prior to spliceosome assembly in vitro.

1992 ◽  
Vol 12 (7) ◽  
pp. 3165-3175 ◽  
Author(s):  
M Bennett ◽  
S Piñol-Roma ◽  
D Staknis ◽  
G Dreyfuss ◽  
R Reed
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Packaging ◽  
Splice Site Selection ◽  
Spliceosome Assembly ◽  
Hnrnp Proteins ◽  
Heterogeneous Nuclear Ribonucleoproteins

We have investigated the composition of the earliest detectable complex (H) assembled on pre-mRNA during the in vitro splicing reaction. We show that most of the proteins in this complex correspond to heterogeneous nuclear ribonucleoproteins (hnRNP), a set of abundant RNA-binding proteins that bind nascent RNA polymerase II transcripts in vivo. Thus, these studies establish a direct parallel between the initial events of RNA processing in vitro and in vivo. In contrast to previous studies, in which total hnRNP particles were isolated from mammalian nuclei, we determined the hnRNP composition of complexes assembled on individual RNAs of defined sequence. We found that a unique combination of hnRNP proteins is associated with each RNA. Thus, our data provide direct evidence for transcript-dependent assembly of pre-mRNA in hnRNP complexes. The observation that pre-mRNA is differentially bound by hnRNP proteins prior to spliceosome assembly suggests the possibility that RNA packaging could play a central role in the mechanism of splice site selection, as well as other posttranscriptional events.

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