scholarly journals Human autoantibody-reactive epitopes of SS-B/La are highly conserved in comparison with epitopes recognized by murine monoclonal antibodies.

1987 ◽  
Vol 166 (6) ◽  
pp. 1627-1640 ◽  
Author(s):  
E K Chan ◽  
E M Tan
Keyword(s):  
Nuclear Protein ◽  
Rna Binding ◽  
Mammalian Species ◽  
Rna Polymerase Iii ◽  
Target Antigen ◽  
Cellular Functions ◽  
Calf Thymus ◽  
Catalytic Sites ◽  
Murine Monoclonal Antibodies ◽  
Murine Mabs

SS-B/La, an ubiquitous nuclear protein of 46-48 kD, is a target antigen of autoantibodies in SLE and Sjogren's syndrome and is involved in the maturation of RNA polymerase III transcripts such as 5S RNA and tRNAs. We have previously shown (14, 15) that SS-B consists of two protease-resistant domains of 23 and 28 kD, with the latter containing the RNA binding site. The epitopes of SS-B/La reactive with human autoantibodies are conserved among several mammalian species examined. BALB/c mice immunized with affinity-purified calf thymus SS-B produce IgG anti-SS-B/La antibodies, which reacted with bovine, human, and rabbit SS-B but not with mouse SS-B/La. The spleen of a mouse with the highest antibody titer was selected for fusion with P3 myeloma. Five IgG1k mAbs (A1-5) were selected by ELISA and immunoblotting. All except A3 reacted with the 28-kD domain. A1, A2, and A3 were capable of immuno-precipitating the 48-kD SS-B protein and its associated RNAs. A1, A2, and A3 also gave fine nuclear speckled staining on human, monkey, bovine, and rabbit cells that was similar in appearance to that with human autoantibodies, but in contrast to staining with human autoantibodies, they did not stain cells from rat, mouse, or rat kangaroo. It appears that human autoantibodies target highly conserved epitopes that can be distinguished from epitopes recognized by immunization-induced murine mAbs. Taken together with other data, it appears that human autoantibodies may be recognizing epitopes that are active or catalytic sites of molecules subserving important cellular functions.

Eukaryotic transcription termination factor La mediates transcript release and facilitates reinitiation by RNA polymerase III

1994 ◽  
Vol 14 (3) ◽  
pp. 2147-2158
Author(s):  
R J Maraia ◽  
D J Kenan ◽  
J D Keene
Keyword(s):  
Rna Polymerase ◽  
Rna Binding ◽  
Transcription Termination ◽  
Rna Polymerase Iii ◽  
Class Iii ◽  
Ample Evidence ◽  
Polymerase Iii ◽  
Transcription Complexes ◽  
Pol Iii

Ample evidence indicates that Alu family interspersed elements retrotranspose via primary transcripts synthesized by RNA polymerase III (pol III) and that this transposition sometimes results in genetic disorders in humans. However, Alu primary transcripts can be processed posttranscriptionally, diverting them away from the transposition pathway. The pol III termination signal of a well-characterized murine B1 (Alu-equivalent) element inhibits RNA 3' processing, thereby stabilizing the putative transposition intermediary. We used an immobilized template-based assay to examine transcription termination by VA1, 7SL, and Alu class III templates and the role of transcript release in the pol III terminator-dependent inhibition of processing of B1-Alu transcripts. We found that the RNA-binding protein La confers this terminator-dependent 3' processing inhibition on transcripts released from the B1-Alu template. Using pure recombinant La protein and affinity-purified transcription complexes, we also demonstrate that La facilitates multiple rounds of transcription reinitiation by pol III. These results illustrate an important role for La in RNA production by demonstrating its ability to clear the termination sites of class III templates, thereby promoting efficient use of transcription complexes by pol III. The role of La as a potential regulatory factor in transcript maturation and how this might apply to Alu interspersed elements is discussed.

scholarly journals An interdomain bridge influences RNA binding of the human La protein

2018 ◽  
Vol 294 (5) ◽  
pp. 1529-1540 ◽  
Author(s):  
Stefano A. Marrella ◽  
Kerene A. Brown ◽  
Farnaz Mansouri-Noori ◽  
Jennifer Porat ◽  
Derek J. Wilson ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Polymerase Iii ◽  
Conformational Dynamics ◽  
Direct Interaction ◽  
Rna Recognition Motif ◽  
Binding Modes ◽  
Nuclear Retention ◽  
Time Resolved ◽  
La Protein ◽  
C Terminus

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.

scholarly journals MONONUCLEOTIDES OF THE CELL NUCLEUS

1957 ◽  
Vol 40 (3) ◽  
pp. 491-513 ◽  
Author(s):  
Syozo Osawa ◽  
V. G. Allfrey ◽  
A. E. Mirsky
Keyword(s):  
Cell Nucleus ◽  
Calcium Ions ◽  
Nuclear Protein ◽  
Adenine Nucleotides ◽  
Close Correlation ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Uridine Diphosphate ◽  
Calf Thymus ◽  
Sucrose Medium

1. It has been demonstrated by ion exchange chromatography that the cell nucleus contains mononucleotides of adenine, guanine, cytosine, uracil, together with diphosphopyridine nucleotide, and several uridine diphosphate derivatives; the adenine nucleotides predominating in amount. Nucleotide components in the cell nucleus are in close agreement both quantitatively and qualitatively with those found in the cytoplasm. 2. In calf thymus sucrose nuclei, nucleotide monophosphates can be phosphorylated to the energy-rich triphosphate form without participation of cytoplasmic components. As to the nature of the phosphorylation, it has been shown that there exist certain differences as well as resemblances between nuclei and mitochondria. A distinctive feature of nuclear phosphorylation is that only intranuclear monophosphates seem to be phosphorylated. The process is completely inhibited by cyanide, azide, and dinitrophenol. However, certain reagents which block oxidative phosphorylation of mitochondria, namely dicumarol, Janus green B, methylene blue, and calcium ions, have no effect on phosphorylation within the nucleus. 3. The bulk of mononucleotides is preserved within thymus nuclei after their isolation in sucrose. Nucleotides are surprisingly well retained by nuclei in a sucrose medium whether or not electrolytes are present and in buffers ranging from pH 3 to 10; under all conditions sucrose is required for retention. 4. Dilute acetate in sucrose releases nucleotides from the nucleus below pH 5.1. As to the effective pH of acetate, there is a sharp boundary between pH 5.1 and pH 5.9. At pH 5.9, and above, acetate does not remove nucleotides from the nucleus. The effects of propionate, formate, and monochloroacetate on the nuclei are the same as that of acetate. 5. When nuclei are exposed to a wide variety of conditions a close correlation is found between the retention in the nucleus of nucleotides and of potassium. This suggests that both substances are part of a common complex in the cell nucleus. 6. It has been shown that upon removal of nucleotides and potassium from calf thymus sucrose nuclei by acetate, the ability to incorporate C14-alanine into nuclear protein is greatly impaired.

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

1999 ◽  
Vol 10 (11) ◽  
pp. 3849-3862 ◽  
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.

scholarly journals The expression of Rpb10, a small subunit common to RNA polymerases, is modulated by the R3H domain-containing Rbs1 protein and the Upf1 helicase

2020 ◽  
Vol 48 (21) ◽  
pp. 12252-12268
Author(s):  
Małgorzata Cieśla ◽  
Tomasz W Turowski ◽  
Marcin Nowotny ◽  
David Tollervey ◽  
Magdalena Boguta
Keyword(s):  
Rna Binding ◽  
Mutational Analysis ◽  
Rna Polymerase Iii ◽  
Protein A ◽  
Small Subunit ◽  
Rna Polymerases ◽  
Genetic Enhancement ◽  
Molecular Approaches ◽  
Regulatory Link ◽  
Pol Iii

Abstract The biogenesis of eukaryotic RNA polymerases is poorly understood. The present study used a combination of genetic and molecular approaches to explore the assembly of RNA polymerase III (Pol III) in yeast. We identified a regulatory link between Rbs1, a Pol III assembly factor, and Rpb10, a small subunit that is common to three RNA polymerases. Overexpression of Rbs1 increased the abundance of both RPB10 mRNA and the Rpb10 protein, which correlated with suppression of Pol III assembly defects. Rbs1 is a poly(A)mRNA-binding protein and mutational analysis identified R3H domain to be required for mRNA interactions and genetic enhancement of Pol III biogenesis. Rbs1 also binds to Upf1 protein, a key component in nonsense-mediated mRNA decay (NMD) and levels of RPB10 mRNA were increased in a upf1Δ strain. Genome-wide RNA binding by Rbs1 was characterized by UV cross-linking based approach. We demonstrated that Rbs1 directly binds to the 3′ untranslated regions (3′UTRs) of many mRNAs including transcripts encoding Pol III subunits, Rpb10 and Rpc19. We propose that Rbs1 functions by opposing mRNA degradation, at least in part mediated by NMD pathway. Orthologues of Rbs1 protein are present in other eukaryotes, including humans, suggesting that this is a conserved regulatory mechanism.

scholarly journals Differential Distribution of RBPMS in Pig, Rat, and Human Retina after Damage

2020 ◽  
Vol 21 (23) ◽  
pp. 9330
Author(s):  
Xandra Pereiro ◽  
Noelia Ruzafa ◽  
J. Haritz Urcola ◽  
Sansar C. Sharma ◽  
Elena Vecino
Keyword(s):  
Rna Binding ◽  
Ganglion Cells ◽  
Mammalian Species ◽  
Plexiform Layer ◽  
Inner Plexiform Layer ◽  
Fiber Layer ◽  
Differential Distribution ◽  
Hypoxic Conditions ◽  
Retinal Explants ◽  
Injury Control

RNA binding protein with multiple splicing (RBPMS) is expressed exclusively in retinal ganglion cells (RGCs) in the retina and can label all RGCs in normal retinas of mice, rats, guinea pigs, rabbits, cats, and monkeys, but its function in these cells is not known. As a result of the limited knowledge regarding RBPMS, we analyzed the expression of RBPMS in the retina of different mammalian species (humans, pigs, and rats), in various stages of development (neonatal and adult) and with different levels of injury (control, hypoxia, and organotypic culture or explants). In control conditions, RBPMS was localized in the RGCs somas in the ganglion cell layer, whereas in hypoxic conditions, it was localized in the RGCs dendrites in the inner plexiform layer. Such differential distributions of RBPMS occurred in all analyzed species, and in adult and neonatal retinas. Furthermore, we demonstrate RBPMS localization in the degenerating RGCs axons in the nerve fiber layer of retinal explants. This is the first evidence regarding the possible transport of RBPMS in response to physiological damage in a mammalian retina. Therefore, RBPMS should be further investigated in relation to its role in axonal and dendritic degeneration.

scholarly journals POLIII-derived non-coding RNAs acting as scaffolds and decoys

2019 ◽  
Vol 11 (10) ◽  
pp. 880-885 ◽  
Author(s):  
Hendrik Täuber ◽  
Stefan Hüttelmaier ◽  
Marcel Köhn
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Current Knowledge ◽  
Cellular Functions ◽  
Control Of Gene Expression ◽  
Ribonucleoprotein Complexes ◽  
Small Ncrnas ◽  
Non Coding Rnas ◽  
And Function

Abstract A large variety of eukaryotic small structured POLIII-derived non-coding RNAs (ncRNAs) have been described in the past. However, for only few, e.g. 7SL and H1/MRP families, cellular functions are well understood. For the vast majority of these transcripts, cellular functions remain unknown. Recent findings on the role of Y RNAs and other POLIII-derived ncRNAs suggest an evolutionarily conserved function of these ncRNAs in the assembly and function of ribonucleoprotein complexes (RNPs). These RNPs provide cellular `machineries’, which are essential for guiding the fate and function of a variety of RNAs. In this review, we summarize current knowledge on the role of POLIII-derived ncRNAs in the assembly and function of RNPs. We propose that these ncRNAs serve as scaffolding factors that `chaperone’ RNA-binding proteins (RBPs) to form functional RNPs. In addition or associated with this role, some small ncRNAs act as molecular decoys impairing the RBP-guided control of RNA fate by competing with other RNA substrates. This suggests that POLIII-derived ncRNAs serve essential and conserved roles in the assembly of larger RNPs and thus the control of gene expression by indirectly guiding the fate of mRNAs and lncRNAs.

Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains

2013 ◽  
Vol 394 (8) ◽  
pp. 1077-1090 ◽  
Author(s):  
Kristin Wächter ◽  
Marcel Köhn ◽  
Nadine Stöhr ◽  
Stefan Hüttelmaier
Keyword(s):  
Subcellular Localization ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Accumulation ◽  
Structural Constraints ◽  
Cellular Functions ◽  
Dependent Protein ◽  
Mrna Binding

Abstract The IGF2 mRNA-binding protein family (IGF2BPs) directs the cytoplasmic fate of various target mRNAs and controls essential cellular functions. The three IGF2BP paralogues expressed in mammals comprise two RNA-recognition motifs (RRM) as well as four KH domains. How these domains direct IGF2BP paralogue-dependent protein function remains largely elusive. In this study, we analyze the role of KH domains in IGF2BPs by the mutational GXXG-GEEG conversion of single KH domain loops in the context of full-length polypeptides. These analyses reveal that all four KH domains of IGF2BP1 and IGF2BP2 are essentially involved in RNA-binding in vitro and the cellular association with RNA-binding proteins (RBPs). Moreover the KH domains prevent the nuclear accumulation of these two paralogues and facilitate their recruitment to stress granules. The role of KH domains appears less pronounced in IGF2BP3, because GxxG-GEEG conversion in all four KH domains only modestly affects RNA-binding, subcellular localization and RNA-dependent protein association of this paralogue. These findings indicate paralogue-dependent RNA-binding properties of IGF2BPs which likely direct distinct cellular functions. Our findings suggest that IGF2BPs contact target RNAs via all four KH domains. This implies significant structural constraints, which presumably allow the formation of exceedingly stable protein-RNA complexes.

scholarly journals Characterization of Conserved Tandem Donor Sites and Intronic Motifs Required for Alternative Splicing in Corticosteroid Receptor Genes

Endocrinology ◽  
2009 ◽  
Vol 150 (11) ◽  
pp. 4958-4967 ◽  
Author(s):  
Caroline Rivers ◽  
Andrea Flynn ◽  
Xiaoxiao Qian ◽  
Laura Matthews ◽  
Stafford Lightman ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Splice Site ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Splice Variants ◽  
Mammalian Species ◽  
Donor Site ◽  
Small Nuclear Rna ◽  
Endogenous Gene ◽  
The One

Alternative splicing events from tandem donor sites result in mRNA variants coding for additional amino acids in the DNA binding domain of both the glucocorticoid (GR) and mineralocorticoid (MR) receptors. We now show that expression of both splice variants is extensively conserved in mammalian species, providing strong evidence for their functional significance. An exception to the conservation of the MR tandem splice site (an A at position +5 of the MR+12 donor site in the mouse) was predicted to decrease U1 small nuclear RNA binding. In accord with this prediction, we were unable to detect the MR+12 variant in this species. The one exception to the conservation of the GR tandem splice site, an A at position +3 of the platypus GRγ donor site that was predicted to enhance binding of U1 snRNA, was unexpectedly associated with decreased expression of the variant from the endogenous gene as well as a minigene. An intronic pyrimidine motif present in both GR and MR genes was found to be critical for usage of the downstream donor site, and overexpression of TIA1/TIAL1 RNA binding proteins, which are known to bind such motifs, led to a marked increase in the proportion of GRγ and MR+12. These results provide striking evidence for conservation of a complex splicing mechanism that involves processes other than stochastic spliceosome binding and identify a mechanism that would allow regulation of variant expression.

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

1997 ◽  
Vol 17 (10) ◽  
pp. 5823-5832 ◽  
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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