scholarly journals Substrate Specificities of SR Proteins in Constitutive Splicing Are Determined by Their RNA Recognition Motifs and Composite Pre-mRNA Exonic Elements

1999 ◽  
Vol 19 (3) ◽  
pp. 1853-1863 ◽  
Author(s):  
Akila Mayeda ◽  
Gavin R. Screaton ◽  
Sharon D. Chandler ◽  
Xiang-Dong Fu ◽  
Adrian R. Krainer
Keyword(s):  
Substrate Specificity ◽  
Sr Proteins ◽  
Specific Substrate ◽  
Rna Recognition ◽  
Negative Effects ◽  
Sr Protein ◽  
Rna Recognition Motifs ◽  
Substrate Specificities ◽  
Constitutive Splicing ◽  
Recognition Motifs

ABSTRACT We report striking differences in the substrate specificities of two human SR proteins, SF2/ASF and SC35, in constitutive splicing. β-Globin pre-mRNA (exons 1 and 2) is spliced indiscriminately with either SR protein. Human immunodeficiency virus tatpre-mRNA (exons 2 and 3) and immunoglobulin μ-chain (IgM) pre-mRNA (exons C3 and C4) are preferentially spliced with SF2/ASF and SC35, respectively. Using in vitro splicing with mutated or chimeric derivatives of the tat and IgM pre-mRNAs, we defined specific combinations of segments in the downstream exons, which mediate either positive or negative effects to confer SR protein specificity. A series of recombinant chimeric proteins consisting of domains of SF2/ASF and SC35 in various combinations was used to localize trans-acting domains responsible for substrate specificity. The RS domains of SF2/ASF and SC35 can be exchanged without effect on substrate specificity. The RNA recognition motifs (RRMs) of SF2/ASF are active only in the context of a two-RRM structure, and RRM2 has a dominant role in substrate specificity. In contrast, the single RRM of SC35 can function alone, but its substrate specificity can be influenced by the presence of an additional RRM. The RRMs behave as modules that, when present in different combinations, can have positive, neutral, or negative effects on splicing, depending upon the specific substrate. We conclude that SR protein-specific recognition of specific positive and negative pre-mRNA exonic elements via one or more RRMs is a crucial determinant of the substrate specificity of SR proteins in constitutive splicing.

scholarly journals A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52.

1997 ◽  
Vol 17 (5) ◽  
pp. 2649-2657 ◽  
Author(s):  
H Shi ◽  
B E Hoffman ◽  
J T Lis
Keyword(s):  
Rna Binding ◽  
Rna Recognition ◽  
Loop Structure ◽  
Hairpin Loop ◽  
Structural Element ◽  
Rna Sequences ◽  
Sr Protein ◽  
Rna Recognition Motifs ◽  
Splicing Regulators ◽  
Recognition Motifs

B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.

scholarly journals RNA splicing specificity determined by the coordinated action of RNA recognition motifs in SR proteins

1997 ◽  
Vol 94 (8) ◽  
pp. 3596-3601 ◽  
Author(s):  
S. D. Chandler ◽  
A. Mayeda ◽  
J. M. Yeakley ◽  
A. R. Krainer ◽  
X.-D. Fu
Keyword(s):  
Rna Splicing ◽  
Sr Proteins ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Coordinated Action ◽  
Recognition Motifs

scholarly journals Nuclear Export and Retention Signals in the RS Domain of SR Proteins

2002 ◽  
Vol 22 (19) ◽  
pp. 6871-6882 ◽  
Author(s):  
Demian Cazalla ◽  
Jun Zhu ◽  
Lisa Manche ◽  
Elisabeth Huber ◽  
Adrian R. Krainer ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Protein Interactions ◽  
Nuclear Export ◽  
Sr Proteins ◽  
Nucleocytoplasmic Shuttling ◽  
Sr Protein ◽  
Nuclear Retention ◽  
Rna Recognition Motifs ◽  
Recognition Motifs

ABSTRACT Splicing factors of the SR protein family share a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal RS domain rich in arginine and serine residues. The RS domain, which is extensively phosphorylated, promotes protein-protein interactions and directs subcellular localization and—in certain situations—nucleocytoplasmic shuttling of individual SR proteins. We analyzed mutant versions of human SF2/ASF in which the natural RS repeats were replaced by RD or RE repeats and compared the splicing and subcellular localization properties of these proteins to those of SF2/ASF lacking the entire RS domain or possessing a minimal RS domain consisting of 10 consecutive RS dipeptides (RS10). In vitro splicing of a pre-mRNA that requires an RS domain could take place when the mutant RD, RE, or RS10 domain replaced the natural domain. The RS10 version of SF2/ASF shuttled between the nucleus and the cytoplasm in the same manner as the wild-type protein, suggesting that a tract of consecutive RS dipeptides, in conjunction with the RRMs of SF2/ASF, is necessary and sufficient to direct nucleocytoplasmic shuttling. However, the SR protein SC35 has two long stretches of RS repeats, yet it is not a shuttling protein. We demonstrate the presence of a dominant nuclear retention signal in the RS domain of SC35.

scholarly journals Isolated pseudo-RNA-recognition motifs of SR proteins can regulate splicing using a noncanonical mode of RNA recognition

2013 ◽  
Vol 110 (30) ◽  
pp. E2802-E2811 ◽  
Author(s):  
A. Clery ◽  
R. Sinha ◽  
O. Anczukow ◽  
A. Corrionero ◽  
A. Moursy ◽  
...  
Keyword(s):  
Sr Proteins ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Recognition Motifs

scholarly journals Structure of the two most C-terminal RNA recognition motifs of PTB using segmental isotope labeling

2005 ◽  
Vol 25 (1) ◽  
pp. 150-162 ◽  
Author(s):  
Francesca Vitali ◽  
Anke Henning ◽  
Florian C Oberstrass ◽  
Yann Hargous ◽  
Sigrid D Auweter ◽  
...  
Keyword(s):  
Isotope Labeling ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Segmental Isotope Labeling ◽  
Recognition Motifs

scholarly journals Specificity of RNA Binding by CPEB: Requirement for RNA Recognition Motifs and a Novel Zinc Finger

1998 ◽  
Vol 18 (2) ◽  
pp. 685-693 ◽  
Author(s):  
Laura E. Hake ◽  
Raul Mendez ◽  
Joel D. Richter
Keyword(s):  
Zinc Finger ◽  
Rna Binding ◽  
Rna Recognition ◽  
Cytoplasmic Polyadenylation ◽  
Functional Motif ◽  
Rna Recognition Motifs ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Maternal Mrnas ◽  
Recognition Motifs

ABSTRACT CPEB is an RNA binding protein that interacts with the maturation-type cytoplasmic polyadenylation element (CPE) (consensus UUUUUAU) to promote polyadenylation and translational activation of maternal mRNAs in Xenopus laevis. CPEB, which is conserved from mammals to invertebrates, is composed of three regions: an amino-terminal portion with no obvious functional motif, two RNA recognition motifs (RRMs), and a cysteine-histidine region that is reminiscent of a zinc finger. In this study, we investigated the physical properties of CPEB required for RNA binding. CPEB can interact with RNA as a monomer, and phosphorylation, which modifies the protein during oocyte maturation, has little effect on RNA binding. Deletion mutations of CPEB have been overexpressed inEscherichia coli and used in a series of RNA gel shift experiments. Although a full-length and a truncated CPEB that lacks 139 amino-terminal amino acids bind CPE-containing RNA avidly, proteins that have had either RRM deleted bind RNA much less efficiently. CPEB that has had the cysteine-histidine region deleted has no detectable capacity to bind RNA. Single alanine substitutions of specific cysteine or histidine residues within this region also abolish RNA binding, pointing to the importance of this highly conserved domain of the protein. Chelation of metal ions by 1,10-phenanthroline inhibits the ability of CPEB to bind RNA; however, RNA binding is restored if the reaction is supplemented with zinc. CPEB also binds other metals such as cobalt and cadmium, but these destroy RNA binding. These data indicate that the RRMs and a zinc finger region of CPEB are essential for RNA binding.

Structural properties and RNA-binding activities of two RNA recognition motifs of a mouse neural RNA-binding protein, mouse-Musashi-1

Gene ◽  
1997 ◽  
Vol 186 (1) ◽  
pp. 21-27 ◽  
Author(s):  
Yasuyuki Kurihara ◽  
Takashi Nagata ◽  
Takao Imai ◽  
Ado Hiwatashi ◽  
Masataka Horiuchi ◽  
...  
Keyword(s):  
Structural Properties ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Recognition Motifs
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