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Binding affinity and cooperativity control U2B?/snRNA/U2A' RNP formation.

ABSTRACT: The U1A and U2B? proteins are components of the U1 and U2 snRNPs, respectively, where they bind to snRNA stemloops. While localization of U1A and U2B? to their respective snRNP is a well-known phenomenon, binding of U2B? to U2 snRNA is typically thought to be accompanied by the U2A' protein. The molecular mechanisms that lead to formation of the RNA/U2B?/U2A' complex and its localization to the U2 snRNP are investigated here, using a combination of in vitro RNA-protein and protein-protein fluorescence and isothermal titration calorimetry binding experiments. We find that U2A' protein binds to U2B? with nanomolar affinity but binds to U1A with only micromolar affinity. In addition, there is RNA-dependent cooperativity (linkage) between protein-protein and protein-RNA binding. The unique combination of tight binding and cooperativity ensures that the U2A'/U2B? complex is partitioned only to the U2 snRNP.

SUBMITTER: Williams SG

PROVIDER: S-EPMC4067145 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Binding affinity and cooperativity control U2B″/snRNA/U2A' RNP formation.

Williams Sandra G SG Hall Kathleen B KB

Biochemistry 20140605 23

The U1A and U2B″ proteins are components of the U1 and U2 snRNPs, respectively, where they bind to snRNA stemloops. While localization of U1A and U2B″ to their respective snRNP is a well-known phenomenon, binding of U2B″ to U2 snRNA is typically thought to be accompanied by the U2A' protein. The molecular mechanisms that lead to formation of the RNA/U2B″/U2A' complex and its localization to the U2 snRNP are investigated here, using a combination of in vitro RNA-protein and protein-protein fluore ...[more]

PMID: 24866816

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Project description:Ribonucleoprotein (RNP) granules transport, store, or degrade messenger RNAs, thereby indirectly regulating protein synthesis. Normally, RNP granules are highly dynamic compartments. However, because of aging or severe environmental stress, RNP granules, in particular stress granules (SGs), convert into solid, aggregate-like inclusions. There is increasing evidence that such RNA-protein inclusions are associated with several age-related neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), fronto-temporal dementia (FTD) and Alzheimer's disease (AD). Thus, understanding what triggers the conversion of RNP granules into aggregates and identifying the cellular players that control RNP granules will be critical to develop treatments for these diseases. In this review article, we discuss recent insight into RNP and SG formation. More specifically, we examine the evidence for liquid-liquid phase separation (LLPS) as an organizing principle of RNP granules and the role of aggregation-prone RNA-binding proteins (RBPs) in this process. We further discuss recent findings that liquid-like SGs can sequester misfolded proteins, which promote an aberrant conversion of liquid SGs into solid aggregates. Importantly, very recent studies show that a specific protein quality control (PQC) process prevents the accumulation of misfolding-prone proteins in SGs and, by doing so, maintains the dynamic state of SGs. This quality control process has been referred to as granulostasis and it relies on the specific action of the HSPB8-BAG3-HSP70 complex. Additional players such as p97/valosin containing protein (VCP) and other molecular chaperones (e.g., HSPB1) participate, directly or indirectly, in granulostasis, and ensure the timely elimination of defective ribosomal products and other misfolded proteins from SGs. Finally, we discuss recent findings that, in the stress recovery phase, SGs are preferentially disassembled with the assistance of chaperones, and we discuss evidence for a back-up system that targets aberrant SGs to the aggresome for autophagy-mediated clearance. Altogether the findings discussed here provide evidence for an intricate network of interactions between RNP granules and various components of the PQC machinery. Molecular chaperones in particular are emerging as key players that control the composition and dynamics of RNP granules, which may be important to protect against age-related diseases.

Project description:The rnp-4f gene in Drosophila melanogaster encodes nuclear protein RNP-4F. This encoded protein is represented by homologs in other eukaryotic species, where it has been shown to function as an intron splicing assembly factor. Here, RNP-4F is believed to initially bind to a recognition sequence on U6-snRNA, serving as a chaperone to facilitate its association with U4-snRNA by intermolecular hydrogen bonding. RNA conformations are a key factor in spliceosome function, so that elucidation of changing secondary structures for interacting snRNAs is a subject of considerable interest and importance. Among the five snRNAs which participate in removal of spliceosomal introns, there is a growing consensus that U6-snRNA is the most structurally dynamic and may constitute the catalytic core. Previous studies by others have generated potential secondary structures for free U4- and U6-snRNAs, including the Y-shaped U4-/U6-snRNA model. These models were based on study of RNAs from relatively few species, and the popular Y-shaped model remains to be systematically re-examined with reference to the many new sequences generated by recent genomic sequencing projects. We have utilized a comparative phylogenetic approach on 60 diverse eukaryotic species, which resulted in a revised and improved U4-/U6-snRNA secondary structure. This general model is supported by observation of abundant compensatory base mutations in every stem, and incorporates more of the nucleotides into base-paired associations than in previous models, thus being more energetically stable. We have extensively sampled the eukaryotic phylogenetic tree to its deepest roots, but did not find genes potentially encoding either U4- or U6-snRNA in the Giardia and Trichomonas data-bases. Our results support the hypothesis that nuclear introns in these most deeply rooted eukaryotes may represent evolutionary intermediates, sharing characteristics of both group II and spliceosomal introns. An unexpected result of this study was discovery of a potential competitive binding site for Drosophila splicing assembly factor RNP-4F to a 5'-UTR regulatory region within its own premRNA, which may play a role in negative feedback control.

Dataset Information

Binding affinity and cooperativity control U2B?/snRNA/U2A' RNP formation.

Publications

Binding affinity and cooperativity control U2B″/snRNA/U2A' RNP formation.

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