Conserved amino acid residues within and outside of the N-terminal ribonucleoprotein motif of U1A small nuclear ribonucleoprotein involved in U1 RNA binding

doi:10.1016/0022-2836(91)90651-L

Journal of Molecular Biology

Volume 219, Issue 4, 20 June 1991, Pages 577-584

https://doi.org/10.1016/0022-2836(91)90651-L Get rights and content

Abstract

By the use of hybrids between a U1 small nuclear ribonucleoprotein (snRNP: U1A) and a U2 snRNP (U2B″) we have identified regions containing 29 U1A-specific amino acid residues scattered throughout the 117 N-terminal residues of the protein, which are involved in binding to U1 RNA. The U1A-specific amino acid residues have been arbitrarily divided into seven contiguous groups. None of these groups is sufficient for U1 binding when transferred singly into the U2B″ context, and none of the groups is essential for U1 binding in U1A. Several different combinations of two or more groups can, however, confer the ability to bind U1 RNA to U2B″, suggesting that most or all of the U1A-specific amino acid residues contribute incrementally to the strength of the specific binding interaction. Further evidence for the importance of the U1A-specific amino acid residues, some of which lie outside the region previously shown to be sufficient for U1 RNA binding, is obtained by comparison of the sequences of human and Xenopus laevis U1A cDNAs. These are extremely similar (94.4% identical) between amino acid residues 7 and 114 but much less conserved immediately upstream and downstream from this region.

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The U1 RNA-binding site of the U1 small nuclear ribonucleo-protein (snRNP)-associated A protein suggests a similarity with U2 snRNPs

Mol. Cell. Biol

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Cited by (44)

Multistep kinetics of the U1A-SL2 RNA complex dissociation
2011, Journal of Molecular Biology
Citation Excerpt :
RRMs bind to single-stranded RNAs of diverse sequence in a variety of structural contexts. Extensive biophysical and biochemical investigations have made the U1A protein a paradigm of RRM–RNA recognition.8,32,33,35–56 U1A is a component of the U1snRNP (U2 small nuclear ribonucleoprotein) and regulates polyadenylation of U1A and other pre-mRNAs.19,57–62
The U1A–SL2 RNA complex is a model system for studying interactions between RNA and the RNA recognition motif (RRM), which is one of the most common RNA binding domains. We report here kinetic studies of dissociation of the U1A–SL2 RNA complex, using laser temperature jump and stopped-flow fluorescence methods with U1A proteins labeled with the intrinsic chromophore tryptophan. An analysis of the kinetic data suggests three phases of dissociation with time scales of ∼ 100 μs, ∼ 50 ms, and ∼ 2 s. We propose that the first step of dissociation is a fast rearrangement of the complex to form a loosely bound complex. The intermediate step is assigned to be the dissociation of the U1A–SL2 RNA complex, and the final step is assigned to a reorganization of the U1A protein structure into the conformation of the free protein. These assignments are consistent with previous proposals based on thermodynamic, NMR, and surface plasmon resonance experiments and molecular dynamics simulations. Together, these results begin to build a comprehensive model of the complex dynamic processes involved in the formation and dissociation of an RRM–RNA complex.
<sup>13</sup>C NMR relaxation studies of RNA base and ribose nuclei reveal a complex pattern of motions in the RNA binding site for human U1A protein
2005, Journal of Molecular Biology
The widespread importance of induced fit and order–disorder transition in RNA recognition by proteins and small molecules makes it imperative that RNA motional properties are characterized quantitatively. Until now, however, very few studies have been dedicated to the systematic characterization of RNA motion and to their changes upon protein or small-molecule binding. The U1A protein–RNA complexes provide some of the best-studied examples of the role of RNA motional changes upon protein binding. Here, we report ¹³C NMR relaxation studies of base and ribose dynamics for the RNA internal loop target of human U1A protein located within the 3′-untranslated region (3′-UTR) of the mRNA coding for U1A itself. We also report the semi-quantitative analysis of both fast (nano- to picosecond) and intermediate (micro- to millisecond) motions for this paradigmatic RNA system. We measure ¹³C T₁, T_1ρ and heteronuclear nuclear Overhauser effects (NOEs) for sugar and base nuclei, as well as the power dependence of T_1ρ at 500 MHz and 750 MHz, and analyze these results using the model-free formalism. The results provide a much clearer picture of the type of motions experienced by this RNA in the absence of the protein than was provided by the analysis of the structure based solely on NOEs and scalar couplings. They define a model where the RNA internal loop region ”breathes” on a micro- to millisecond timescale with respect to the double-helical regions. Superimposed on this slower motion, the residues at the very tip of the loop undergo faster (nano- to picosecond) motions. We hypothesize that these motions allow the RNA to sample multiple conformations so that the protein can select a structure within the ensemble that optimizes intermolecular contacts.
Changes in side-chain and backbone dynamics identify determinants of specificity in RNA recognition by human U1A protein
1999, Journal of Molecular Biology
The ribonucleoprotein (RNP) domain is one of the most common eukaryotic protein domains, and is found in many proteins involved in recognition of a wide variety of RNAs. Two structures of RNA complexes of human U1A protein have revealed important aspects of RNP-RNA recognition, but have also raised intriguing questions concerning how RNP domains discriminate between different RNAs. In this work, we extend the investigation of U1A-RNA recognition by comparing the dynamics of U1A protein both free and in complex with RNA. We have also investigated the trimolecular complex between two U1A proteins and the complete polyadenylation inhibition element to study the effect of RNA-dependent protein-protein interactions on protein conformational flexibility. We report that changes in backbone dynamics upon complex formation identify regions of the protein where conformational exchange processes are quenched in the RNA-bound conformation. Furthermore, amino acids whose side-chains experience significant changes in conformational flexibility coincide with residues particularly important for the specificity of the U1A protein/RNA interaction. This study adds a new dimension to the description of the coordinated changes in structure and dynamics that are critical to define the biological specificity of U1A and other RNP proteins.
A novel zinc finger-containing RNA-binding protein conserved from fruitflies to humans
1997, Genomics
TheDrosophila larkgene encodes an essential RNA-binding protein of the RNA recognition motif (RRM) class that is required during embryonic development. Genetic analysis demonstrates that it also functions as a molecular element of a circadian clock output pathway, mediating the temporal regulation of adult emergence in the fruitfly. We now report the molecular characterization of a human gene with significant similarity tolark.Based on fluorescencein situhybridization and radiation hybrid mapping, the human gene has been localized to chromosome region 11q13; it is closely linked to several identified genes including the locus of Bardet–Biedl syndrome type 1. Thelark-homologous human gene expresses a single 1.8-kb size class of mRNA in most or all tissues including brain. Additional database searches have identified a mouse counterpart that is virtually identical to the human protein. Similar to lark protein, both mammalian proteins contain two copies of the RRM-type consensus RNA-binding motif. Unlike most RRM family members, however, theDrosophilaand mammalian proteins also contain a retroviral-type (RT) zinc finger that is situated 43 residues C-terminal to the second RRM element. Within a 184-residue segment spanning the RRM elements and the RT zinc finger, the human and mouse proteins are 61% similar to theDrosophilalark sequence. These common sequence features and comparisons among a large collection of RRM proteins suggest that the human and mouse proteins represent homologues ofDrosophilalark.
Solution structure of the N-terminal RNP domain of U1A protein: The role of C-terminal residues in structure stability and RNA binding
1996, Journal of Molecular Biology
The solution structure of a fragment of the human U1A spliceosomal protein containing residues 2 to 117 (U1A117) determined using multi-dimensional heteronuclear NMR is presented. The C-terminal region of the molecule is considerably more ordered in the free protein than thought previously and its conformation is different from that seen in the crystal structure of the complex with U1 RNA hairpin II. The residues between Asp90 and Lys98 form an α-helix that lies across the β-sheet, with residues Ile93, Ile94 and Met97 making contacts with Leu44, Phe56 and Ile58. This interaction prevents solvent exposure of hydrophobic residues on the surface of the β-sheet, thereby stabilising the protein. Upon RNA binding, helix C moves away from this position, changing its orientation by 135° to allow Tyr13, Phe56 and Gln54 to stack with bases of the RNA, and also allowing Leu44 to contact the RNA. The new position of helix C in the complex with RNA is stabilised by hydrophobic interactions from Ile93 and Ile94 to Ile58, Leu 41, Val62 and His10, as well as a hydrogen bond between Ser91 and Thr11. The movement of helix C mainly involves changes in the main-chain torsion angles of Thr89, Asp90 and Ser91, the helix thereby acting as a "lid" over the RNA binding surface.
Individual RNA recognition motifs of TIA-1 and TIAR have different RNA binding specificities
1996, Journal of Biological Chemistry
TIA-1 and TIAR are two closely related RNA recognition motif (RRM) proteins which possess three RRM-type RNA binding domains (RRMs 1, 2, and 3). Although both proteins have been implicated as effectors of apoptotic cell death, the specific functions of TIA-1 and TIAR are not known. We have performed in vitro selection/amplification from pools of random RNA sequences to identify RNAs to which TIA-1 and TIAR bind with high affinity. Both proteins selected RNAs containing one or several short stretches of uridylate residues suggesting that the two proteins have similar RNA binding specificities. Replacement of the uridylate stretch with an equal number of cytidine residues eliminates the protein-RNA interaction. Mutational analysis indicates that, for both TIA-1 and TIAR, it is the second RNA binding domain (RRM 2) which mediates the specific binding to uridylate-rich RNAs. Although RRM 2 is both necessary and sufficient for this interaction, the affinity for the selected RNA (as determined by filter binding assays) does increase when the second domain of TIAR is expressed together with the first and third domains (K_d = 2 × 10^-8M) rather than alone (K_d = 5 × 10^-8M). Although RRM 3 (of either TIA-1 or TIAR) does not interact with the uridylate-rich sequences selected by the full-length proteins, it is a bona fide RNA binding domain capable of affinity-precipitating a population of cellular RNAs ranging in size from 0.5 to 5 kilobases. In contrast, RRM 1 does not affinity-precipitate cellular RNA. The inability of RRM 1 to interact with RNA may be due to the presence of negatively charged amino acids within the RNP 1 octamer.

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^☆: The work in Holland was carried out under the auspices of the Netherlands Foundation for Chemical Research (SON) and with the financial aid of the Netherlands Organization for Scientific Research (NWO) and the Dutch Rheumatism Foundation. The work in EMBL was supported by EMBO and EMBL.

^†: Present address: University of Geneva, Department of Microbiology, Faculty of Medicine (C.M.U), Av. de Champel, 1211 Geneva 4, Switzerland.

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References (22)

A binding consensus: RNA-protein interactions in splicing, snRNPs and sex

Cell

mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence

Mol. Cell. Biol

Structure-probing of U1 snRNPs gradually depleted of the U1-specific proteins A, C and 70K. Evidence that A interacts differentially with developmentally regulated mouse U1 snRNA variants

Nucl. Acids Res

RNA-binding proteins as developmental regulators

Genes Develop

A weak interaction between the U2A′ protein and U2 snRNA helps to stabilize their complex with the U2B″ protein

Nucl. Acids Res

Structure and expression of a Xenopus gene encoding an snRNP protein (U1 70K)

EMBO J

Leucine periodicity of U2 small nuclear ribonucleo-protein particle (snRNP) A′ protein is implicated in snRNP assembly via protein-protein interactions

Mol. Cell. Biol

Spliceosomal snRNAs

Annu. Rev. Genet

Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B″ antigen

snRNP proteins

The U1 RNA-binding site of the U1 small nuclear ribonucleo-protein (snRNP)-associated A protein suggests a similarity with U2 snRNPs

Mol. Cell. Biol

Cited by (44)

Multistep kinetics of the U1A-SL2 RNA complex dissociation

<sup>13</sup>C NMR relaxation studies of RNA base and ribose nuclei reveal a complex pattern of motions in the RNA binding site for human U1A protein

Changes in side-chain and backbone dynamics identify determinants of specificity in RNA recognition by human U1A protein

A novel zinc finger-containing RNA-binding protein conserved from fruitflies to humans

Solution structure of the N-terminal RNP domain of U1A protein: The role of C-terminal residues in structure stability and RNA binding

Individual RNA recognition motifs of TIA-1 and TIAR have different RNA binding specificities

Journal of Molecular Biology

CommunicationConserved amino acid residues within and outside of the N-terminal ribonucleoprotein motif of U1A small nuclear ribonucleoprotein involved in U1 RNA binding☆

Abstract

Cell

mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence

Mol. Cell. Biol

Structure-probing of U1 snRNPs gradually depleted of the U1-specific proteins A, C and 70K. Evidence that A interacts differentially with developmentally regulated mouse U1 snRNA variants

Nucl. Acids Res

RNA-binding proteins as developmental regulators

Genes Develop

A weak interaction between the U2A′ protein and U2 snRNA helps to stabilize their complex with the U2B″ protein

Nucl. Acids Res

Structure and expression of a Xenopus gene encoding an snRNP protein (U1 70K)

EMBO J

Leucine periodicity of U2 small nuclear ribonucleo-protein particle (snRNP) A′ protein is implicated in snRNP assembly via protein-protein interactions

Mol. Cell. Biol

Spliceosomal snRNAs

Annu. Rev. Genet

Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B″ antigen

snRNP proteins

The U1 RNA-binding site of the U1 small nuclear ribonucleo-protein (snRNP)-associated A protein suggests a similarity with U2 snRNPs

Mol. Cell. Biol

Communication
Conserved amino acid residues within and outside of the N-terminal ribonucleoprotein motif of U1A small nuclear ribonucleoprotein involved in U1 RNA binding☆