RESUMEN
Numerous RNA binding proteins are deposited onto an mRNA transcript to modulate post-transcriptional processing events ensuring proper mRNA maturation. Defining the interplay between RNA binding proteins that couple mRNA biogenesis events is crucial for understanding how gene expression is regulated. To explore how RNA binding proteins control mRNA processing, we investigated a role for the evolutionarily conserved polyadenosine RNA binding protein, Nab2, in mRNA maturation within the nucleus. This work reveals that nab2 mutant cells accumulate intron-containing pre-mRNA in vivo We extend this analysis to identify genetic interactions between mutant alleles of nab2 and genes encoding the splicing factor, MUD2, and the RNA exosome, RRP6, with in vivo consequences of altered pre-mRNA splicing and poly(A) tail length control. As further evidence linking Nab2 proteins to splicing, an unbiased proteomic analysis of vertebrate Nab2, ZC3H14, identifies physical interactions with numerous components of the spliceosome. We validated the interaction between ZC3H14 and U2AF2/U2AF65 Taking all the findings into consideration, we present a model where Nab2/ZC3H14 interacts with spliceosome components to allow proper coupling of splicing with subsequent mRNA processing steps contributing to a kinetic proofreading step that allows properly processed mRNA to exit the nucleus and escape Rrp6-dependent degradation.
RESUMEN
Ribosomal protein S4 binds and stabilizes a five-helix junction or five-way junction (5WJ) in the 5' domain of 16S ribosomal RNA (rRNA) and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and 5WJ reorganize their structures. We show that labile S4 complexes rearrange into stable complexes within a few minutes at 42 °C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show that this structural change depends only on 16S residues within the S4 binding site. SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension) chemical probing experiments showed that S4 strongly stabilizes 5WJ and the helix (H) 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in H16 that makes specific contacts with the S4 N-terminal extension, as well as a right-angle motif between H3, H4, and H18, requires a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of H3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation.
Asunto(s)
Escherichia coli/metabolismo , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Temperatura , Factores de TiempoRESUMEN
Mutations in genes associated with the U4/U6-U5 small nuclear ribonucleoprotein (snRNP) complex of the spliceosome are implicated in autosomal-dominant retinitis pigmentosa (adRP), a group of progressive retinal degenerative disorders leading to visual impairment, loss of visual field, and even blindness. We recently assigned a locus (RP33) for adRP to 2cen-q12.1, a region that harbors the SNRNP200 gene encoding hBrr2, another U4/U6-U5 snRNP component that is required for unwinding of U4/U6 snRNAs during spliceosome activation and for disassembly of the spliceosome. Here, we report the identification of a missense mutation, c.3260C>T (p.S1087L), in exon 25 of the SNRNP200 gene in an RP33-linked family. The c.3260C>T substitution showed complete cosegregation with the retinitis pigmentosa (RP) phenotype over four generations, but was absent in a panel of 400 controls. The p.S1087L mutation and p.R1090L, another adRP-associated allele, reside in the "ratchet" helix of the first of two Sec63 domains implicated in the directionality and processivity of nucleic acid unwinding. Indeed, marked defects in U4/U6 unwinding, but not U4/U6-U5 snRNP assembly, were observed in budding yeast for the analogous mutations (N1104L and R1107L) of the corresponding Brr2p residues. The linkage of hBrr2 to adRP suggests that the mechanism of pathogenesis for splicing-factor-related RP may fundamentally derive from a defect in hBrr2-dependent RNA unwinding and a consequent defect in spliceosome activation.
Asunto(s)
Mutación , Retinitis Pigmentosa/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Ribonucleoproteína Nuclear Pequeña U4-U6/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Exones , Femenino , Genes Dominantes , Marcadores Genéticos , Células HeLa , Humanos , Leucina/metabolismo , Escala de Lod , Masculino , Repeticiones de Microsatélite , Datos de Secuencia Molecular , Mutación Missense , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Linaje , Mapeo Físico de Cromosoma , Empalme del ARN , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteína Nuclear Pequeña U5 , Homología de Secuencia de AminoácidoRESUMEN
Primary ribosomal protein S4 is essential for 30S ribosome biogenesis in eubacteria, because it nucleates subunit assembly and helps coordinate assembly with the synthesis of its rRNA and protein components. S4 binds a five-helix junction (5WJ) that bridges the 5' and 3' ends of the 16S 5' domain. To delineate which nucleotides contribute to S4 recognition, sequential deletions of the 16S 5' domain were tested in competitive S4-binding assays based on electrophoretic mobility shifts. S4 binds the minimal 5WJ RNA containing just the five-helix junction as well or better than with affinity comparable to or better than the 5' domain or native 16S rRNA. Internal deletions and point mutations demonstrated that helices 3, 4, 16 and residues at the helix junctions are necessary for S4 binding, while the conserved helix 18 pseudoknot is dispensable. Hydroxyl radical footprinting and chemical base modification showed that S4 makes the same interactions with minimal rRNA substrates as with the native 16S rRNA, but the minimal substrates are more pre-organized for binding S4. Together, these results suggest that favorable interactions with S4 offset the energetic penalty for folding the 16S rRNA.
Asunto(s)
ARN Ribosómico 16S/química , Proteínas Ribosómicas/química , Sitios de Unión , Unión Competitiva , Escherichia coli/genética , Unión Proteica , Huella de Proteína , Estructura Terciaria de Proteína , ARN Ribosómico 16S/metabolismo , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismoRESUMEN
Rapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. How the Escherichia coli 16S ribosomal RNA and the 20 proteins that make up the 30S ribosomal subunit can assemble correctly in a few minutes remains a challenging problem, partly because of the lack of real-time data on the earliest stages of assembly. By providing snapshots of individual RNA and protein interactions as they emerge in real time, here we show that 30S assembly nucleates concurrently from different points along the rRNA. Time-resolved hydroxyl radical footprinting was used to map changes in the structure of the rRNA within 20 milliseconds after the addition of total 30S proteins. Helical junctions in each domain fold within 100 ms. In contrast, interactions surrounding the decoding site and between the 5', the central and the 3' domains require 2-200 seconds to form. Unexpectedly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA-protein encounter complexes refold during assembly. Although early steps in assembly are linked to intrinsically stable rRNA structure, later steps correspond to regions of induced fit between the proteins and the rRNA.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Conformación de Ácido Nucleico , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Escherichia coli/química , Cinética , Modelos Moleculares , Unión Proteica , Conformación Proteica , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/química , Sincrotrones , Rayos XRESUMEN
Endothelial cells (EC) express both hypoxia inducible factor-1alpha (HIF-1alpha) and -2alpha (HIF-2alpha), yet their roles in the EC hypoxic response are unclear. Hypoxia upregulates the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EC through a 5' hypoxic regulatory element (HRE). We compared the upregulation of GAPDH in human lung microvascular EC to that in hep3B cells, another cell type known to express both HIF-1alpha and HIF-2alpha. GAPDH mRNA increased to a lesser extent in hypoxic hep3B cells than in EC, yet upregulation occurred through the same HRE that was active in EC. HIF-1alpha protein induction in response to hypoxia was similar in both cell types. In contrast, HIF-2alpha protein levels were upregulated to a greater extent and for a longer period of time by hypoxia in EC than in hep3B cells. Correspondingly, electrophoretic mobility supershift assays showed that, in EC, there was preferential binding of HIF-2alpha to the GAPDH HRE while, in hep3B cells, there was binding of both HIF-1alpha and HIF-2alpha. The preferential binding of HIF-2alpha to the GAPDH HRE in EC may account for their higher level of induction of GAPDH. These findings suggest that cell-specific patterns of HIF-1alpha and HIF-2alpha expression lead to cell-specific gene upregulation during hypoxia.