Molecular basis of RNA recognition and TAP binding by the SR proteins SRp20 and 9G8

ANE syndrome is a ribosomopathy caused by a mutation in an RNA recognition motif of RBM28, a nucleolar protein conserved to yeast (Nop4). While patients with ANE syndrome have fewer mature ribosomes, it is unclear how this mutation disrupts ribosome assembly. Here we use yeast as a model system and show that the mutation confers growth and pre-rRNA processing defects. Recently, we found that Nop4 is a hub protein in the nucleolar large subunit (LSU) processome interactome. Here we demonstrate that the ANE syndrome mutation disrupts Nop4’s hub function by abrogating several of Nop4’s protein-protein interactions. Circular dichroism and NMR demonstrate that the ANE syndrome mutation in RRM3 of human RBM28 disrupts domain folding. We conclude that the ANE syndrome mutation generates defective protein folding which abrogates protein-protein interactions and causes faulty pre-LSU rRNA processing, thus revealing one aspect of the molecular basis of this human disease.

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Structural and dynamic studies of the human RNA binding protein RBM3 reveals the molecular basis of its oligomerization and RNA recognition

FEBS Journal ◽

10.1111/febs.16301 ◽

2021 ◽

Author(s):

Sayantani Roy ◽

Soumendu Boral ◽

Snigdha Maiti ◽

Tushar Kushwaha ◽

Aditya J. Basak ◽

...

Keyword(s):

Molecular Basis ◽

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Rna Recognition ◽

Dynamic Studies

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Molecular Basis for Target RNA Recognition and Cleavage by Human RISC

Cell ◽

10.1016/j.cell.2007.04.037 ◽

2007 ◽

Vol 130 (1) ◽

pp. 101-112 ◽

Cited By ~ 332

Author(s):

Stefan Ludwig Ameres ◽

Javier Martinez ◽

Renée Schroeder

Keyword(s):

Molecular Basis ◽

Rna Recognition ◽

Target Rna

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Molecular basis of UG-rich RNA recognition by the human splicing factor TDP-43

Nature Structural & Molecular Biology ◽

10.1038/nsmb.2698 ◽

2013 ◽

Vol 20 (12) ◽

pp. 1443-1449 ◽

Cited By ~ 141

Author(s):

Peter J Lukavsky ◽

Dalia Daujotyte ◽

James R Tollervey ◽

Jernej Ule ◽

Cristiana Stuani ◽

...

Keyword(s):

Molecular Basis ◽

Splicing Factor ◽

Rna Recognition

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Human SR proteins and isolation of a cDNA encoding SRp75

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4023-4028.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4023-4028

Author(s):

A M Zahler ◽

K M Neugebauer ◽

J A Stolk ◽

M B Roth

Keyword(s):

Monoclonal Antibody ◽

Amino Acid ◽

Active Sites ◽

Sr Proteins ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Rich Region ◽

Recognition Motif ◽

S100 Extract ◽

Arginine Residues

SR proteins are a family of proteins that have a common epitope recognized by a monoclonal antibody (MAb104) that binds active sites of polymerase II transcription. Four of the SR family members have been shown to restore activity to an otherwise splicing-deficient extract (S100 extract). Here we show that two untested SR proteins, SRp20 and SRp75, can also complement the splicing-deficient extract. We isolated a cDNA encoding SRp75 and found that this protein, like other SR proteins, contains an N-terminal RNA recognition motif (RRM), a glycine-rich region, an internal region homologous to the RRM, and a long (315-amino-acid) C-terminal domain composed predominantly of alternating serine and arginine residues. The apparent molecular mass of dephosphorylated SRp75 is 57 kDa, the size predicted from the cDNA clone. We also detected mobility shifts after dephosphorylating SRp55, SRp40, SRp30a, and SRp30b; the sizes of the shifts are proportional to the length of the SR domain, suggesting that serines in this domain are phosphorylated.

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Human SR proteins and isolation of a cDNA encoding SRp75.

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4023 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4023-4028 ◽

Cited By ~ 57

Author(s):

A M Zahler ◽

K M Neugebauer ◽

J A Stolk ◽

M B Roth

Keyword(s):

Monoclonal Antibody ◽

Amino Acid ◽

Active Sites ◽

Sr Proteins ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Rich Region ◽

Recognition Motif ◽

S100 Extract ◽

Arginine Residues

SR proteins are a family of proteins that have a common epitope recognized by a monoclonal antibody (MAb104) that binds active sites of polymerase II transcription. Four of the SR family members have been shown to restore activity to an otherwise splicing-deficient extract (S100 extract). Here we show that two untested SR proteins, SRp20 and SRp75, can also complement the splicing-deficient extract. We isolated a cDNA encoding SRp75 and found that this protein, like other SR proteins, contains an N-terminal RNA recognition motif (RRM), a glycine-rich region, an internal region homologous to the RRM, and a long (315-amino-acid) C-terminal domain composed predominantly of alternating serine and arginine residues. The apparent molecular mass of dephosphorylated SRp75 is 57 kDa, the size predicted from the cDNA clone. We also detected mobility shifts after dephosphorylating SRp55, SRp40, SRp30a, and SRp30b; the sizes of the shifts are proportional to the length of the SR domain, suggesting that serines in this domain are phosphorylated.

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Regulation of Alternative Splicing by SRrp86 and Its Interacting Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.23.21.7437-7447.2003 ◽

2003 ◽

Vol 23 (21) ◽

pp. 7437-7447 ◽

Cited By ~ 28

Author(s):

Jun Li ◽

Ian C. Hawkins ◽

Christopher D. Harvey ◽

Jennifer L. Jennings ◽

Andrew J. Link ◽

...

Keyword(s):

Alternative Splicing ◽

Sr Proteins ◽

Splicing Factors ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Protein Activity ◽

Splice Site Selection ◽

Sr Protein ◽

Library Screen ◽

Specific Proteins

ABSTRACT SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.

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

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.1853 ◽

1999 ◽

Vol 19 (3) ◽

pp. 1853-1863 ◽

Cited By ~ 85

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.

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