scholarly journals PAB1 Self-Association Precludes Its Binding to Poly(A), Thereby Accelerating CCR4 Deadenylation In Vivo

2007 ◽  
Vol 27 (17) ◽  
pp. 6243-6253 ◽  
Author(s):  
Gang Yao ◽  
Yueh-Chin Chiang ◽  
Chongxu Zhang ◽  
Darren J. Lee ◽  
Thomas M. Laue ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Interactions ◽  
Rna Binding ◽  
Decay Rates ◽  
Mrna Turnover ◽  
Rich Region ◽  
Binding Surface ◽  
Self Association ◽  
Proline Rich Region

ABSTRACT The mRNA deadenylation process, catalyzed by the CCR4 deadenylase, is known to be the major factor controlling mRNA decay rates in Saccharomyces cerevisiae. We have identified the proline-rich region and RRM1 domains of poly(A) binding protein (PAB1) as necessary for CCR4 deadenylation. Deletion of either of these regions but not other regions of PAB1 significantly reduced PAB1-PAB1 protein interactions, suggesting that PAB1 oligomerization is a required step for deadenylation. Moreover, defects in these two regions inhibited the formation of a novel, circular monomeric PAB1 species that forms in the absence of poly(A). Removal of the PAB1 RRM3 domain, which promoted PAB1 oligomerization and circularization, correspondingly accelerated CCR4 deadenylation. Circular PAB1 was unable to bind poly(A), and PAB1 multimers were severely deficient or unable to bind poly(A), implicating the PAB1 RNA binding surface as critical in making contacts that allow PAB1 self-association. These results support the model that the control of CCR4 deadenylation in vivo occurs in part through the removal of PAB1 from the poly(A) tail following its self-association into multimers and/or a circular species. Known alterations in the P domains of different PAB proteins and factors and conditions that affect PAB1 self-association would, therefore, be expected to be critical to controlling mRNA turnover in the cell.

scholarly journals A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

1996 ◽  
Vol 16 (2) ◽  
pp. 548-556 ◽  
Author(s):  
N L Freeman ◽  
T Lila ◽  
K A Mintzer ◽  
Z Chen ◽  
A J Pahk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Adenylyl Cyclase ◽  
Sh3 Domain ◽  
Regulatory Function ◽  
Nucleotide Exchange ◽  
Cortical Actin ◽  
Rich Region ◽  
Sh3 Domains ◽  
Proline Rich Region

Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.

scholarly journals Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

2021 ◽  
Vol 4 (1) ◽  
pp. 22
Author(s):  
Mrinmoyee Majumder ◽  
Viswanathan Palanisamy
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Control Of Gene Expression ◽  
Regulatory Pathways ◽  
Advantages And Disadvantages ◽  
Protein Nucleic Acid

Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

scholarly journals RNA-Centric Approaches to Profile the RNA–Protein Interaction Landscape on Selected RNAs

2021 ◽  
Vol 7 (1) ◽  
pp. 11 ◽  
Author(s):  
André P. Gerber
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Regulatory Networks ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Protein Complexes ◽  
Cell Protein ◽  
Transcriptional Regulatory Networks ◽  
Technological Advances

RNA–protein interactions frame post-transcriptional regulatory networks and modulate transcription and epigenetics. While the technological advances in RNA sequencing have significantly expanded the repertoire of RNAs, recently developed biochemical approaches combined with sensitive mass-spectrometry have revealed hundreds of previously unrecognized and potentially novel RNA-binding proteins. Nevertheless, a major challenge remains to understand how the thousands of RNA molecules and their interacting proteins assemble and control the fate of each individual RNA in a cell. Here, I review recent methodological advances to approach this problem through systematic identification of proteins that interact with particular RNAs in living cells. Thereby, a specific focus is given to in vivo approaches that involve crosslinking of RNA–protein interactions through ultraviolet irradiation or treatment of cells with chemicals, followed by capture of the RNA under study with antisense-oligonucleotides and identification of bound proteins with mass-spectrometry. Several recent studies defining interactomes of long non-coding RNAs, viral RNAs, as well as mRNAs are highlighted, and short reference is given to recent in-cell protein labeling techniques. These recent experimental improvements could open the door for broader applications and to study the remodeling of RNA–protein complexes upon different environmental cues and in disease.

A Proline-Rich Region in the Zeste Protein Essential for Transvection and white Repression by Zeste1

Genetics ◽  
1998 ◽  
Vol 148 (4) ◽  
pp. 1865-1874
Author(s):  
Christina Rosen ◽  
Dale Dorsett ◽  
Joseph Jack
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Homologous Chromosome ◽  
Protein Complexes ◽  
Dna Binding Protein ◽  
Polycomb Group ◽  
The Self ◽  
Rich Region ◽  
Interaction Domain ◽  
Proline Rich Region

Abstract The DNA-binding protein encoded by the zeste gene of Drosophila activates transcription and mediates interchromosomal interactions such as transvection. The mutant protein encoded by the zeste1 (z1) allele retains the ability to support transvection, but represses white. Similar to transvection, repression requires Zeste-Zeste protein interactions and a second copy of white, either on the homologous chromosome or adjacent on the same chromosome. We characterized two pseudorevertants of z1 (z1-35 and z1-42) and another zeste mutation (z78c) that represses white. The z1 lesion alters a lysine residue located between the N-terminal DNA-binding domain and the C-terminal hydrophobic repeats involved in Zeste self-interactions. The z78c mutation alters a histidine near the site of the z1 lesion. Both z1 pseudorevertants retain the z1 lesion and alter different prolines in a proline-rich region located between the z1 lesion and the self-interaction domain. The pseudorevertants retain the ability to self-interact, but fail to repress white or support transvection at Ultrabithorax. To account for these observations and evidence indicating that Zeste affects gene expression through Polycomb group (Pc-G) protein complexes that epigenetically maintain chromatin states, we suggest that the regions affected by the z1, z78c, and pseudorevertant lesions mediate interactions between Zeste and the maintenance complexes.

scholarly journals PRIMA: a gene-centered, RNA-to-protein method for mapping RNA-protein interactions

2016 ◽  
Author(s):  
Alex M. Tamburino ◽  
Ebru Kaymak ◽  
Shaleen Shrestha ◽  
Amy D. Holdorf ◽  
Sean P. Ryder ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Small Rna ◽  
Rna Binding ◽  
Mrna Translation ◽  
Single Experiment ◽  
Novel Interactions ◽  
Transcriptional Gene Regulation ◽  
Rna Interaction ◽  
Eukaryotic Genomes

SUMMARYInteractions between RNA binding protein (RBP) and mRNAs are critical to post-transcriptional gene regulation. Eukaryotic genomes encode thousands of mRNAs and hundreds of RBPs. However, in contrast to interactions between transcription factors (TFs) and DNA, the interactome between RBPs and RNA has been explored for only a small number of proteins and RNAs. This is largely because the focus has been on using ‘protein-centered’ (RBP-to-RNA) interaction mapping methods that identify the RNAs with which an individual RBP interacts. While powerful, these methods cannot as of yet be applied to the entire RBPome. Moreover, it may be desirable for a researcher to identify the repertoire of RBPs that can interact with an mRNA of interest – in a ‘gene-centered’ manner, yet few such techniques are available. Here, we present Protein-RNA Interaction Mapping Assay (PRIMA) with which an RNA ‘bait’ can be tested versus multiple RBP ‘preys’ in a single experiment. PRIMA is a translation-based assay that examines interactions in the yeast cytoplasm, the cellular location of mRNA translation. We show that PRIMA can be used with small RNA elements, as well as with full-length Caenorhabditis elegans 3′UTRs. PRIMA faithfully recapitulates numerous well-characterized RNA-RBP interactions and also identified novel interactions, some of which were confirmed in vivo. We envision that PRIMA will provide a complementary tool to expand the depth and scale with which the RNA-RBP interactome can be explored.

RNA binding by Sxl proteins in vitro and in vivo

1994 ◽  
Vol 14 (7) ◽  
pp. 4975-4990
Author(s):  
M E Samuels ◽  
D Bopp ◽  
R A Colvin ◽  
R F Roscigno ◽  
M A Garcia-Blanco ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Target Genes ◽  
Rna Binding ◽  
Polytene Chromosomes ◽  
Regulatory Sequences ◽  
Acceptor Site ◽  
Nuclear Extracts ◽  
Preferential Binding

Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sxl protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sxl was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat puffs following shock.

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.

ZNF198–FGFR1 transforming activity depends on a novel proline-rich ZNF198 oligomerization domain

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 699-704 ◽  
Author(s):  
Sheng Xiao ◽  
Jennifer G. McCarthy ◽  
Jon C. Aster ◽  
Jonathan A. Fletcher
Keyword(s):  
Tyrosine Kinase ◽  
Growth Factor Receptor ◽  
Chromosomal Translocation ◽  
Rich Region ◽  
Kinase Activation ◽  
Distinctive Type ◽  
Transforming Activity ◽  
Self Association ◽  
Proline Rich Region ◽  
Oligomerization Domain

Abstract An acquired chromosomal translocation, t(8;13)(p11;q11-12), observed in a distinctive type of stem cell leukemia/lymphoma syndrome, leads to the fusion of the 5′ portion of ZNF198 and the 3′ portion of FGFR1. ZNF198–FGFR1 fusion transcripts encode 4 to 10 zinc fingers, a proline-rich region, and the intracellular portion of the FGFR1 (fibroblast growth factor receptor 1) receptor tyrosine kinase. We demonstrate that the ZNF198 proline-rich region constitutes a novel self-association domain. When fused to the intracellular domain of FGFR1, the ZNF198 proline-rich region is sufficient to cause oligomerization, FGFR1 tyrosine kinase activation, and transformation of Ba/F3 cells to IL-3 independent growth.

ZNF198–FGFR1 transforming activity depends on a novel proline-rich ZNF198 oligomerization domain

Blood ◽  
2000 ◽  
Vol 96 (2) ◽  
pp. 699-704 ◽  
Author(s):  
Sheng Xiao ◽  
Jennifer G. McCarthy ◽  
Jon C. Aster ◽  
Jonathan A. Fletcher
Keyword(s):  
Tyrosine Kinase ◽  
Growth Factor Receptor ◽  
Chromosomal Translocation ◽  
Rich Region ◽  
Kinase Activation ◽  
Distinctive Type ◽  
Transforming Activity ◽  
Self Association ◽  
Proline Rich Region ◽  
Oligomerization Domain

An acquired chromosomal translocation, t(8;13)(p11;q11-12), observed in a distinctive type of stem cell leukemia/lymphoma syndrome, leads to the fusion of the 5′ portion of ZNF198 and the 3′ portion of FGFR1. ZNF198–FGFR1 fusion transcripts encode 4 to 10 zinc fingers, a proline-rich region, and the intracellular portion of the FGFR1 (fibroblast growth factor receptor 1) receptor tyrosine kinase. We demonstrate that the ZNF198 proline-rich region constitutes a novel self-association domain. When fused to the intracellular domain of FGFR1, the ZNF198 proline-rich region is sufficient to cause oligomerization, FGFR1 tyrosine kinase activation, and transformation of Ba/F3 cells to IL-3 independent growth.

scholarly journals Extracellular Signal-Regulated Kinase 1 Interacts with and Phosphorylates CdGAP at an Important Regulatory Site

2005 ◽  
Vol 25 (15) ◽  
pp. 6314-6329 ◽  
Author(s):  
Joseph Tcherkezian ◽  
Eric I. Danek ◽  
Sarah Jenna ◽  
Ibtissem Triki ◽  
Nathalie Lamarche-Vane
Keyword(s):  
Bound States ◽  
Mitogen Activated Protein Kinase ◽  
Regulatory Site ◽  
Nucleotide Exchange ◽  
Rich Region ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Proline Rich Region

ABSTRACT Rho GTPases regulate multiple cellular processes affecting both cell proliferation and cytoskeletal dynamics. Their cycling between inactive GDP- and active GTP-bound states is tightly regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We have previously identified CdGAP (for Cdc42 GTPase-activating protein) as a specific GAP for Rac1 and Cdc42. CdGAP consists of an N-terminal RhoGAP domain and a C-terminal proline-rich region. In addition, CdGAP is a member of the impressively large number of mammalian RhoGAP proteins that is well conserved among both vertebrates and invertebrates. In mice, we find two predominant isoforms of CdGAP differentially expressed in specific tissues. We report here that CdGAP is highly phosphorylated in vivo on serine and threonine residues. We find that CdGAP is phosphorylated downstream of the MEK-extracellular signal-regulated kinase (ERK) pathway in response to serum or platelet-derived growth factor stimulation. Furthermore, CdGAP interacts with and is phosphorylated by ERK-1 and RSK-1 in vitro. A putative DEF (docking for ERK FXFP) domain located in the proline-rich region of CdGAP is required for efficient binding and phosphorylation by ERK1/2. We identify Thr776 as an in vivo target site of ERK1/2 and as an important regulatory site of CdGAP activity. Together, these data suggest that CdGAP is a novel substrate of ERK1/2 and mediates cross talk between the Ras/mitogen-activated protein kinase pathway and regulation of Rac1 activity.

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