The Evolutionarily-conserved Polyadenosine RNA Binding Protein, Nab2, Cooperates with Splicing Machinery to Regulate the Fate of pre-mRNA

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/U2AF^(65). 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

A minimized rRNA-binding site for ribosomal protein S4 and its implications for 30S assembly

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

Evolutionarily conserved polyadenosine RNA binding protein Nab2 cooperates with splicing machinery to regulate the fate of pre-mRNA

Numerous RNA binding proteins are deposited onto an mRNA transcript to modulate posttranscriptional 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 study 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 a splicing factor, MUD2, and 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/U2AF(65). 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

Autosomal-Dominant Retinitis Pigmentosa Caused by a Mutation in SNRNP200, a Gene Required for Unwinding of U4/U6 snRNAs

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