SummaryRapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. 1,2 How the 16S rRNA and 20 proteins that make up the 30S ribosomal subunit assemble faithfully in a few minutes remains a challenging problem, in part because real-time data on the earliest stages of assembly are lacking. Here, we show that 30S assembly nucleates concurrently from different points along the rRNA, by providing snapshots of individual RNA and protein interactions as they emerge in real time. Time-resolved hydroxyl radical footprinting 3 was used to map changes in the structure of the rRNA within 20 ms after addition of total 30S proteins. Helix junctions in each domain fold within 100 ms. By contrast, interactions surrounding the decoding site and between the 5′, central and 3′ domains require 2-200 seconds to form. Surprisingly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA-protein encounter complexes refold during assembly. While 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.Nomura and colleagues demonstrated that hierarchical addition of ribosomal proteins to the 16S rRNA produces cooperativity, 4 that is due to protein-induced structural changes in the 16S rRNA rather than direct contacts between proteins. 5 As the rRNA becomes more structured as proteins join the complex, assembly is coupled to the folding pathway of the rRNA. 6,7 The simplest kinetic model for 30S assembly is that regions of the 16S rRNA contacted by primary assembly proteins fold earliest, while helices stabilized by tertiary assembly proteins fold last. If assembly is strictly sequential, each subdomain of the rRNA will fold within a distinct time, producing a limited set of intermediate complexes.Alternatively, the rate of protein binding may initially depend on the stochastic probability of forming locally stable rRNA and protein interactions, with progression to later intermediates depending on propagation of this conformational order to neighboring regions in the rRNA. If more than one region of the naked rRNA can fold, assembly is expected to nucleate from many places at once, producing an ensemble of reconstitution intermediates and multi-stage assembly kinetics. 8 *Corresponding author: E-mail: swoodson@jhu.edu, tel. +001-410-516-2015, FAX +001-410-516-4118 To visualize the intermediates of 30S ribosome assembly, the structure of the 16S rRNA was probed by time-resolved X-ray hydroxyl radical footprinting (Figure 1). The extent of RNA cleavage correlates with backbone exposure. 9 Thus, this method probes individual tertiary contacts in the rRNA as well as protein interactions that bury the rRNA backbone. Previous efforts to map the conformational changes in the 16S rRNA during 30S ribosome assembly used low temperature or subsets of proteins to stall assembly at specific stages. 10,11 We took advantage of the...

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.

g 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. Gene expression is temporally and spatially regulated to produce a precise protein expression profile that dictates the function of each cell. Although much of this control occurs at the level of transcription, both co-and posttranscriptional events also play key regulatory roles. Newly synthesized mRNAs undergo numerous processing events, including 5= capping, splicing, 3=-end processing, and export to the cytoplasm (1, 2). Ensuring the synchrony of mRNA biogenesis requires RNA binding proteins that not only perform the processing tasks but also couple the events to ensure that only properly processed mRNAs are available for translation in the cytoplasm (3).Key processing events that must be coordinated include splicing and 3=-end processing. Although steps in mRNA processing are often depicted and studied as separate events, there is a growing body of evidence that these processing events are intimately coupled to one another (2). For example, splicing and 3=-end processing are coupled in humans as mutations in splice site and polyadenylation consensus sequences mutually disrupt both splicing and polyadenylation (4-6). In addition, a number of splicing factors copurify with the 3=-end processing complex (7-10). For example, there is evidence that the splicing factor U2AF2/ U2AF 65 functions as a bridge between the U2 snRNP and the 3=-end processing machinery (11,12). Given the extensive coupling between RNA processing steps, it is important to consider the consequences when one step of the process is disrupted in vivo. Cells have developed numerous overlapping mechanisms to ensure that faulty mRNA transcripts are not...

Ribosomal protein S4 binds and stabilizes a five-helix junction in the 5’ domain of the 16S rRNA, and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and the five-helix junction reorganize their structures. We show that labile S4 complexes rearrange to 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 this structural change depends only on 16S residues within the S4 binding site. SHAPE chemical-probing experiments showed that S4 strongly stabilizes the five-helix junction and helix 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in helix 16 that makes specific contacts with the S4 N-terminal extension, and a right angle motif between helices 3, 4 and 18, require a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of helix 3, 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.

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.