scholarly journals The Stem-Loop Binding Protein (SLBP1) Is Present in Coiled Bodies of the Xenopus Germinal Vesicle

1999 ◽  
Vol 10 (2) ◽  
pp. 487-499 ◽  
Author(s):  
Jennifer Abbott ◽  
William F. Marzluff ◽  
Joseph G. Gall
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Germinal Vesicle ◽  
Xenopus Laevis Oocytes ◽  
Coiled Body ◽  
Stem Loop ◽  
Subnuclear Localization ◽  
Small Nuclear Ribonucleoprotein ◽  
Coiled Bodies ◽  
U7 Snrnp

The stem-loop binding protein (SLBP1) binds the 3′ stem-loop of histone pre-mRNA and is required for efficient processing of histone transcripts in the nucleus. We examined the localization of SLBP1 in the germinal vesicle of Xenopus laevis oocytes. In spread preparations of germinal vesicle contents, an anti-SLBP1 antibody stained coiled bodies and specific chromosomal loci, including terminal granules, axial granules, and some loops. After injection ofmyc-tagged SLBP1 transcripts into the oocyte cytoplasm, newly translated myc-SLBP1 protein was detectable in coiled bodies within 4 h and in terminal and axial granules by 8 h. To identify the region(s) of SLBP1 necessary for subnuclear localization, we subcloned various parts of the SLBP1 cDNA and injected transcripts of these into the cytoplasm of oocytes. We determined that 113 amino acids at the carboxy terminus of SLBP1 are sufficient for coiled body localization and that disruption of a previously defined RNA-binding domain did not alter this localization. Coiled bodies also contain the U7 small nuclear ribonucleoprotein particle (snRNP), which participates in cleavage of the 3′ end of histone pre-mRNA. The colocalization of SLBP1 and the U7 snRNP in the coiled body suggests coordinated control of their functions, perhaps through a larger histone-processing particle. Some coiled bodies are attached to the lampbrush chromosomes at the histone gene loci, consistent with the view that coiled bodies in the oocyte recruit histone-processing factors to the sites of histone pre-mRNA transcription. The non-histone chromosomal sites at which SLBP1 is found include the genes coding for 5 S rRNA, U1 snRNA, and U2 snRNA, suggesting a wider role for SLBP1 in the biosynthesis of small non-spliced RNAs.

scholarly journals Stem-Loop Binding Protein Facilitates 3′-End Formation by Stabilizing U7 snRNP Binding to Histone Pre-mRNA

1999 ◽  
Vol 19 (5) ◽  
pp. 3561-3570 ◽  
Author(s):  
Zbigniew Dominski ◽  
Lian-Xing Zheng ◽  
Ricardo Sanchez ◽  
William F. Marzluff
Keyword(s):  
Amino Acids ◽  
Rna Binding ◽  
Binding Protein ◽  
Nuclear Extract ◽  
Stable Complex ◽  
Stem Loop ◽  
Terminal Domain ◽  
C Terminus ◽  
Loop Sequence ◽  
U7 Snrnp

ABSTRACT The 3′ end of histone mRNA is formed by an endonucleolytic cleavage of the primary transcript after a conserved stem-loop sequence. The cleavage reaction requires at least two trans-acting factors: the stem-loop binding protein (SLBP), which binds the stem-loop sequence, and the U7 snRNP that interacts with a sequence downstream from the cleavage site. Removal of SLBP from a nuclear extract abolishes 3′-end processing, and the addition of recombinant SLBP restores processing activity of the depleted extract. To determine the regions of human SLBP necessary for 3′ processing, various deletion mutants of the protein were tested for their ability to complement the SLBP-depleted extract. The entire N-terminal domain and the majority of the C-terminal domain of human SLBP are dispensable for processing. The minimal protein that efficiently supports cleavage of histone pre-mRNA consists of 93 amino acids containing the 73-amino-acid RNA-binding domain and 20 amino acids located immediately next to its C terminus. Replacement of these 20 residues with an unrelated sequence in the context of the full-length SLBP reduces processing >90%. Coimmunoprecipitation experiments with the anti-SLBP antibody demonstrated that SLBP and U7 snRNP form a stable complex only in the presence of pre-mRNA substrates containing a properly positioned U7 snRNP binding site. One role of SLBP is to stabilize the interaction of the histone pre-mRNA with U7 snRNP.

scholarly journals Mutations in the RNA Binding Domain of Stem-Loop Binding Protein Define Separable Requirements for RNA Binding and for Histone Pre-mRNA Processing

2001 ◽  
Vol 21 (6) ◽  
pp. 2008-2017 ◽  
Author(s):  
Zbigniew Dominski ◽  
Judith A. Erkmann ◽  
John A. Greenland ◽  
William F. Marzluff
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Binding Activity ◽  
Mrna Processing ◽  
Binding Domain ◽  
Stem Loop ◽  
Amino Acid Region ◽  
Stem Loop Structure ◽  
U7 Snrnp ◽  
Rna Binding Domain

ABSTRACT Expression of replication-dependent histone genes at the posttranscriptional level is controlled by stem-loop binding protein (SLBP). One function of SLBP is to bind the stem-loop structure in the 3′ untranslated region of histone pre-mRNAs and facilitate 3′ end processing. Interaction of SLBP with the stem-loop is mediated by the centrally located RNA binding domain (RBD). Here we identify several highly conserved amino acids in the RBD mutation of which results in complete or substantial loss of SLBP binding activity. We also identify residues in the RBD which do not contribute to binding to the stem-loop RNA but instead are required for efficient recruitment of U7 snRNP to histone pre-mRNA. Recruitment of the U7 snRNP to the pre-mRNA also depends on the 20-amino-acid region located immediately downstream of the RBD. A critical region of the RBD contains the sequence YDRY. The tyrosines are required for RNA binding, and the DR dipeptide is essential for processing but not for RNA binding. It is likely that the RBD of SLBP interacts directly with both the stem-loop RNA and other processing factor(s), most likely the U7 snRNP, to facilitate histone pre-mRNA processing.

scholarly journals The RNA-binding protein Sam68 modulates the alternative splicing of Bcl-x

2007 ◽  
Vol 176 (7) ◽  
pp. 929-939 ◽  
Author(s):  
Maria Paola Paronetto ◽  
Tilman Achsel ◽  
Autumn Massiello ◽  
Charles E. Chalfant ◽  
Claudio Sette
Keyword(s):  
Alternative Splicing ◽  
Tyrosine Phosphorylation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Live Cells ◽  
Hnrnp A1 ◽  
Splice Site Selection ◽  
Subnuclear Localization ◽  
Mrna Targets

The RNA-binding protein Sam68 is involved in apoptosis, but its cellular mRNA targets and its mechanism of action remain unknown. We demonstrate that Sam68 binds the mRNA for Bcl-x and affects its alternative splicing. Depletion of Sam68 by RNA interference caused accumulation of antiapoptotic Bcl-x(L), whereas its up-regulation increased the levels of proapoptotic Bcl-x(s). Tyrosine phosphorylation of Sam68 by Fyn inverted this effect and favored the Bcl-x(L) splice site selection. A point mutation in the RNA-binding domain of Sam68 influenced its splicing activity and subnuclear localization. Moreover, coexpression of ASF/SF2 with Sam68, or fusion with an RS domain, counteracted Sam68 splicing activity toward Bcl-x. Finally, Sam68 interacted with heterogenous nuclear RNP (hnRNP) A1, and depletion of hnRNP A1 or mutations that impair this interaction attenuated Bcl-x(s) splicing. Our results indicate that Sam68 plays a role in the regulation of Bcl-x alternative splicing and that tyrosine phosphorylation of Sam68 by Src-like kinases can switch its role from proapoptotic to antiapoptotic in live cells.

A cDNA clone for a polyadenylated RNA-binding protein of Xenopus laevis oocytes hybridizes to four developmentally regulated mRNAs

1985 ◽  
Vol 5 (10) ◽  
pp. 2697-2704
Author(s):  
L J Lorenz ◽  
J D Richter
Keyword(s):  
Xenopus Laevis ◽  
Cdna Clone ◽  
Rna Binding ◽  
Binding Protein ◽  
Single Gene ◽  
In Vitro Translation ◽  
Xenopus Laevis Oocytes ◽  
Cdna Insert ◽  
Alternative Processing

Xenopus laevis oocytes contain a unique group of proteins which decrease during oogenesis, bind poly(A) RNA, and possibly play a role in the regulation of translation. A monoclonal antibody generated against one of these proteins was used to screen an expression vector cDNA library. A cDNA clone was isolated and confirmed to code for the binding protein by in vitro translation of hybrid-selected RNA followed by immunoprecipitation. This cDNA, when used in RNA gel blots, hybridized to four transcripts of 2.0, 1.7 (two transcripts of similar size), and 1.2 kilobases. All of the transcripts decreased in amount during oogenesis and were not evident in somatic cells. In addition, the fraction of the transcripts associated with polysomes decreased during oogenesis. Digestion of the cDNA insert with PstI generated two fragments of 220 and 480 base pairs which, when used as probes in an RNA gel blot, hybridized to unique as well as common transcripts. Genomic Southern blots suggested the presence of a single gene, indicating that these transcripts arose by alternative processing.

scholarly journals Distinct Poly(rC) Binding Protein KH Domain Determinants for Poliovirus Translation Initiation and Viral RNA Replication

2002 ◽  
Vol 76 (23) ◽  
pp. 12008-12022 ◽  
Author(s):  
Brandon L. Walter ◽  
Todd B. Parsley ◽  
Ellie Ehrenfeld ◽  
Bert L. Semler
Keyword(s):  
Translation Initiation ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Replication ◽  
Viral Rna ◽  
Host Protein ◽  
Loop Structure ◽  
Stem Loop ◽  
Stem Loop Structure

ABSTRACT The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5′-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.

scholarly journals NMR structure and dynamics of the RNA-binding site for the histone mRNA stem-loop binding protein

RNA ◽  
2002 ◽  
Vol 8 (1) ◽  
pp. 83-96 ◽  
Author(s):  
ERIC S. DEJONG ◽  
WILLIAM F. MARZLUFF ◽  
EDWARD P. NIKONOWICZ
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Binding Protein ◽  
Nmr Structure ◽  
Histone Mrna ◽  
Stem Loop ◽  
Structure And Dynamics

scholarly journals U7 snRNP is recruited to histone pre-mRNA in a FLASH-dependent manner by two separate regions of the stem–loop binding protein

RNA ◽  
2017 ◽  
Vol 23 (6) ◽  
pp. 938-951 ◽  
Author(s):  
Aleksandra Skrajna ◽  
Xiao-cui Yang ◽  
Katarzyna Bucholc ◽  
Jun Zhang ◽  
Traci M. Tanaka Hall ◽  
...  
Keyword(s):  
Binding Protein ◽  
Dependent Manner ◽  
Stem Loop ◽  
U7 Snrnp

scholarly journals U6atac snRNA stem-loop interacts with U12 p65 RNA binding protein and is functionally interchangeable with the U12 apical stem-loop III

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Jagjit Singh ◽  
Kavleen Sikand ◽  
Heike Conrad ◽  
Cindy L. Will ◽  
Anton A. Komar ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Stem Loop

scholarly journals Polypyrimidine tract-binding protein blocks miRNA-124 biogenesis to enforce its neuronal-specific expression in the mouse

2018 ◽  
Vol 115 (47) ◽  
pp. E11061-E11070 ◽  
Author(s):  
Kyu-Hyeon Yeom ◽  
Simon Mitchell ◽  
Anthony J. Linares ◽  
Sika Zheng ◽  
Chia-Ho Lin ◽  
...  
Keyword(s):  
Neuronal Differentiation ◽  
Rna Binding ◽  
Binding Protein ◽  
Embryonic Stem ◽  
Specific Expression ◽  
Stem Loop ◽  
Polypyrimidine Tract Binding Protein ◽  
Precursor Rna

MicroRNA (miRNA)-124 is expressed in neurons, where it represses genes inhibitory for neuronal differentiation, including the RNA binding protein PTBP1. PTBP1 maintains nonneuronal splicing patterns of mRNAs that switch to neuronal isoforms upon neuronal differentiation. We find that primary (pri)-miR-124-1 is expressed in mouse embryonic stem cells where mature miR-124 is absent. PTBP1 binds to this precursor RNA upstream of the miRNA stem–loop to inhibit mature miR-124 expression in vivo and DROSHA cleavage of pri-miR-124-1 in vitro. This function for PTBP1 in repressing miR-124 biogenesis defines an additional regulatory loop in the already intricate interplay between these two molecules. Applying mathematical modeling to examine the dynamics of this regulation, we find that the pool of pri-miR-124 whose maturation is blocked by PTBP1 creates a robust and self-reinforcing transition in gene expression as PTBP1 is depleted during early neuronal differentiation. While interlocking regulatory loops are often found between miRNAs and transcriptional regulators, our results indicate that miRNA targeting of posttranscriptional regulators also reinforces developmental decisions. Notably, induction of neuronal differentiation observed upon PTBP1 knockdown likely results from direct derepression of miR-124, in addition to indirect effects previously described.

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