Faculty Opinions recommendation of Structural analysis of cooperative RNA binding by the La motif and central RRM domain of human La protein.

La proteins are RNA chaperones that perform various functions depending on distinct RNA-binding modes and their subcellular localization. In the nucleus, they help process UUU-3′OH–tailed nascent RNA polymerase III transcripts, such as pre-tRNAs, whereas in the cytoplasm they contribute to translation of poly(A)-tailed mRNAs. La accumulation in the nucleus and cytoplasm is controlled by several trafficking elements, including a canonical nuclear localization signal in the extreme C terminus and a nuclear retention element (NRE) in the RNA recognition motif 2 (RRM2) domain. Previous findings indicate that cytoplasmic export of La due to mutation of the NRE can be suppressed by mutations in RRM1, but the mechanism by which the RRM1 and RRM2 domains functionally cooperate is poorly understood. In this work, we use electromobility shift assays (EMSA) to show that mutations in the NRE and RRM1 affect binding of human La to pre-tRNAs but not UUU-3′OH or poly(A) sequences, and we present compensatory mutagenesis data supporting a direct interaction between the RRM1 and RRM2 domains. Moreover, we use collision-induced unfolding and time-resolved hydrogen–deuterium exchange MS analyses to study the conformational dynamics that occur when this interaction is intact or disrupted. Our results suggest that the intracellular distribution of La may be linked to its RNA-binding modes and provide the first evidence for a direct protein–protein interdomain interaction in La proteins.

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Sla1, a Schizosaccharomyces pombe Homolog of the Human La Protein, Induces Ectopic Meiosis when Its C Terminus Is Truncated

Eukaryotic Cell ◽

10.1128/ec.2.6.1274-1287.2003 ◽

2003 ◽

Vol 2 (6) ◽

pp. 1274-1287 ◽

Cited By ~ 9

Author(s):

Kaori Tanabe ◽

Noriko Ito ◽

Tomomi Wakuri ◽

Fumiyo Ozoe ◽

Makoto Umeda ◽

...

Keyword(s):

Schizosaccharomyces Pombe ◽

Rna Binding ◽

Rna Binding Proteins ◽

Small Region ◽

Full Length ◽

Rna Recognition Motifs ◽

La Protein ◽

C Terminus ◽

Localization Signal ◽

Recognition Motifs

ABSTRACT Sla1 is a Schizosaccharomyces pombe homolog of the human La protein. La proteins are known to be RNA-binding proteins that bear conserved RNA recognition motifs (La and RRMs), but their biological functions still have not been fully resolved. In this study, we show that the S. pombe La homolog (Sla1) is involved in regulating sexual development. Sla1 truncated in the C terminus (Sla1ΔC) induced ectopic sporulation in the ras1Δ strain and several other sporulation-deficient mutants. The C terminus contains a nuclear localization signal. While full-length Sla1 localizes in the nucleus, Sla1ΔC is found throughout the cell, suggesting the cytoplasmic localization of Sla1ΔC is involved in its sporulation-inducing activity. Further deletion analysis of Sla1 indicated that a small region (35 amino acids) that includes a portion of RRM2 is sufficient to induce sporulation. The La motif (RRM1) is not involved in this activity. Strikingly, Sla1ΔC induced haploid meiosis in a heterothallic strain, similar to the pat1-114 or mei2-SATA mutation. Sla1ΔC induced sporulation in a mei3 disruptant but not in a mei2 disruptant, indicating that Sla1ΔC requires Mei2 to induce haploid meiosis. Deletion of the chromosomal sla1 gene lowered the temperature sensitivity of the pat1-114 mutant. Two-hybrid analysis indicated that Pat1 interacts with Sla1ΔC but not full-length Sla1. Thus, Sla1ΔC may block Pat1 activity. This block would remove the inhibition on Mei2, which would then drive the cell into haploid meiosis. Finally, Sla1 was degraded prior to the start of meiosis when we monitored Sla1 in cells in which meiosis was synchronously induced. The ability of truncated Sla1 to induce ectopic meiosis represents a very novel function that has hitherto not been suspected for the La family of proteins.

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NMR Structural Study of TcUBP1, a Single RRM Domain Protein fromTrypanosoma cruzi: Contribution of a β Hairpin to RNA Binding†,‡

Biochemistry ◽

10.1021/bi047450e ◽

2005 ◽

Vol 44 (10) ◽

pp. 3708-3717 ◽

Cited By ~ 25

Author(s):

Laurent Volpon ◽

Iván D'Orso ◽

Christopher R. Young ◽

Alberto C. Frasch ◽

Kalle Gehring

Keyword(s):

Structural Study ◽

Rna Binding ◽

Rrm Domain ◽

Domain Protein

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Two Yeast La Motif-containing Proteins Are RNA-binding Proteins that Associate with Polyribosomes

Molecular Biology of the Cell ◽

10.1091/mbc.10.11.3849 ◽

1999 ◽

Vol 10 (11) ◽

pp. 3849-3862 ◽

Cited By ~ 36

Author(s):

Suzanne G. Sobel ◽

Sandra L. Wolin

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Polymerase Iii ◽

Mrna Translation ◽

Protein Synthesis Inhibitors ◽

La Protein ◽

Evolutionarily Conserved ◽

Synthetically Lethal ◽

Type Strains

We have characterized two Saccharomyces cerevisiaeproteins, Sro9p and Slf1p, which contain a highly conserved motif found in all known La proteins. Originally described as an autoantigen in patients with rheumatic disease, the La protein binds to newly synthesized RNA polymerase III transcripts. In yeast, the La protein homologue Lhp1p is required for the normal pathway of tRNA maturation and also stabilizes newly synthesized U6 RNA. We show that deletions in both SRO9 and SLF1 are not synthetically lethal with a deletion in LHP1, indicating that the three proteins do not function in a single essential process. Indirect immunofluorescence microscopy reveals that although Lhp1p is primarily localized to the nucleus, Sro9p is cytoplasmic. We demonstrate that Sro9p and Slf1p are RNA-binding proteins that associate preferentially with translating ribosomes. Consistent with a role in translation, strains lacking either Sro9p or Slf1p are less sensitive than wild-type strains to certain protein synthesis inhibitors. Thus, Sro9p and Slf1p define a new and possibly evolutionarily conserved class of La motif-containing proteins that may function in the cytoplasm to modulate mRNA translation.

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Structural basis underlying CAC RNA recognition by the RRM domain of dimeric RNA-binding protein RBPMS

Quarterly Reviews of Biophysics ◽

10.1017/s0033583515000207 ◽

2015 ◽

Vol 49 ◽

Cited By ~ 21

Author(s):

Marianna Teplova ◽

Thalia A. Farazi ◽

Thomas Tuschl ◽

Dinshaw J. Patel

Keyword(s):

Rna Binding ◽

Binding Protein ◽

Rna Binding Protein ◽

Hek293 Cells ◽

Structural Basis ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Binding Studies ◽

Muscle Plasticity ◽

Rrm Domain

AbstractRNA-binding protein with multiple splicing (designated RBPMS) is a higher vertebrate mRNA-binding protein containing a single RNA recognition motif (RRM). RBPMS has been shown to be involved in mRNA transport, localization and stability, with key roles in axon guidance, smooth muscle plasticity, as well as regulation of cancer cell proliferation and migration. We report on structure-function studies of the RRM domain of RBPMS bound to a CAC-containing single-stranded RNA. These results provide insights into potential topologies of complexes formed by the RBPMS RRM domain and the tandem CAC repeat binding sites as detected by photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation. These studies establish that the RRM domain of RBPMS forms a symmetrical dimer in the free state, with each monomer binding sequence-specifically to all three nucleotides of a CAC segment in the RNA bound state. Structure-guided mutations within the dimerization and RNA-binding interfaces of RBPMS RRM on RNA complex formation resulted in both disruption of dimerization and a decrease in RNA-binding affinity as observed by size exclusion chromatography and isothermal titration calorimetry. As anticipated from biochemical binding studies, over-expression of dimerization or RNA-binding mutants of Flag-HA-tagged RBPMS were no longer able to track with stress granules in HEK293 cells, thereby documenting the deleterious effects of such mutations in vivo.

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Anti-La Monoclonal Antibodies Recognizing Epitopes Within the RNA-Binding Domain of the La Protein Show Differential Capacities to Immunoprecipitate RNA-Associated La Protein

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1995.611zz.x ◽

1995 ◽

Vol 232 (2) ◽

pp. 611-619 ◽

Cited By ~ 32

Author(s):

Ger J. M. Pruijn ◽

Jose P. H. Thijssen ◽

Paul R. Smith ◽

David G. Williams ◽

Walther J. Venrooij

Keyword(s):

Monoclonal Antibodies ◽

Rna Binding ◽

Binding Domain ◽

La Protein ◽

Rna Binding Domain

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Identification and Structural Analysis of Human RBM8A and RBM8B: Two Highly Conserved RNA-Binding Motif Proteins That Interact with OVCA1, a Candidate Tumor Suppressor

Genomics ◽

10.1006/geno.2000.6315 ◽

2000 ◽

Vol 69 (1) ◽

pp. 54-62 ◽

Cited By ~ 28

Author(s):

Ana M. Salicioni ◽

Mingrong Xi ◽

Lisa A. Vanderveer ◽

Binaifer Balsara ◽

Joseph R. Testa ◽

...

Keyword(s):

Structural Analysis ◽

Tumor Suppressor ◽

Rna Binding ◽

Binding Motif ◽

Candidate Tumor Suppressor

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A carboxy-terminal basic region controls RNA polymerase III transcription factor activity of human La protein.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.5823 ◽

1997 ◽

Vol 17 (10) ◽

pp. 5823-5832 ◽

Cited By ~ 51

Author(s):

J L Goodier ◽

H Fan ◽

R J Maraia

Keyword(s):

Transcription Factor ◽

Rna Polymerase ◽

Rna Binding ◽

Rna Polymerase Iii ◽

Transcription Factor Activity ◽

Factor Activity ◽

Terminal Domain ◽

La Protein ◽

Pol Iii ◽

Basic Region

Human La protein has been shown to serve as a transcription factor for RNA polymerase III (pol III) by facilitating transcription termination and recycling of transcription complexes. In addition, La binds to the 3' oligo(U) ends common to all nascent pol III transcripts, and in the case of B1-Alu RNA, protects it from 3'-end processing (R. J. Maraia, D. J. Kenan, and J. D. Keene, Mol. Cell. Biol. 14:2147-2158, 1994). Others have previously dissected the La protein into an N-terminal domain that binds RNA and a C-terminal domain that does not. Here, deletion and substitution mutants of La were examined for general RNA binding, RNA 3'-end protection, and transcription factor activity. Although some La mutants altered in a C-terminal basic region bind RNA in mobility shift assays, they are defective in RNA 3'-end protection and do not support transcription, while one C-terminal substitution mutant is defective only in transcription. Moreover, a C-terminal fragment lacking RNA binding activity appears able to support low levels of transcription by pol III. While efficient multiround transcription is supported only by mutants that bind RNA and contain a C-terminal basic region. These analyses indicate that RNA binding contributes to but is not sufficient for La transcription factor activity and that the C-terminal domain plays a role in transcription that is distinguishable from simple RNA binding. The transcription factor activity of La can be reversibly inhibited by RNA, suggesting the potential for feedback inhibition of pol III transcription.

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