tRNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments (tRFs)) are an abundant class of small non-coding RNAs whose biological roles are not well defined. We show that inhibition of a specific tsRNA, LeuCAG3′tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (for RPS28 and RPS15) to enhance their translation. Reduction of RPS28 mRNA translation blocks pre-18S ribosomal RNA processing, resulting in a decrease in the number of 40S ribosomal subunits. These data establish another post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.

Ribosomal protein S5 is critical for small ribosomal subunit (SSU) assembly and is indispensable for SSU function. Previously, we identified a point mutation in S5, (G28D) that alters both SSU formation and translational fidelity in vivo, which is unprecedented for other characterized S5 mutations. Surprisingly, additional copies of an extraribosomal assembly factor, RimJ, rescued all the phenotypes associated with S5(G28D), including fidelity defects, suggesting that the effect of RimJ on rescuing the miscoding of S5(G28D) is indirect. To understand the underlying mechanism, we focused on the biogenesis cascade and observed defects in processing of precursor 16S (p16S) rRNA in the S5(G28D) strain, which were rescued by RimJ. Analyses of p16S rRNA-containing ribosomes from other strains further supported a correspondence between the extent of 5 0 end maturation of 16S rRNA and translational miscoding. Chemical probing of mutant ribosomes with additional leader sequences at the 5 0 end of 16S rRNA compared to WT ribosomes revealed structural differences in the region of helix 1. Thus, the presence of additional nucleotides at the 5 0 end of 16S rRNA could alter fidelity by changing the architecture of 16S rRNA in translating ribosomes and suggests that fidelity is governed by accuracy and completeness of the SSU biogenesis cascade.R-protein S5 | ram mutations | ribosome biogenesis | 16S rRNA processing O ne of the most remarkable feats of the ribosome is the ability to decode genetic information accurately in a process that involves the interaction of aminoacyl-transfer RNAs (aa-tRNAs) to the aa-tRNA binding site (A site) on the small ribosomal subunit (SSU; 30S). However, this process is not fully error proof and missense errors occur at a frequency of 10 −3 to 10 −4 per amino acid synthesized (1). Decoding is thought to involve a number of local and global conformational changes in the SSU upon binding of a cognate aa-tRNA to the A site. These structural changes result in a transition from the "open" to the "closed" form whereby the head of the SSU rotates toward the shoulder and the shoulder toward the platform (2). The r-proteins S4, S5, and S12 along with helices 27 and 44 of 16S rRNA are implicated in fidelity; mutations in S4 and S5 can lead to ribosome ambiguity (ram) or miscoding, whereas specific mutations in S12 lead to hyperaccuracy (3). Based on the recent crystal structures (2), the r-protein ram mutations map at the interface of S4 and S5 and disrupt a number of salt bridges that are present in the open SSUs. These changes could destabilize the open state, thereby perturbing the equilibrium to promote the closed state and allowing decreased discrimination in the decoding process (4). Some additional biochemical and structural data support this model (5); nonetheless, other data are hard to incorporate into this scheme. A few mutations in S4 can confer "restrictive" phenotypes to Salmonella typhimurium (6) and surprisingly these hyperaccurate alleles of S4 suppress the hyperaccurate phenotypes of S1...

S5 is a small subunit ribosomal protein (r-protein) linked to the functional center of the 30S ribosomal subunit. In this study we have identified a unique amino acid mutation in Escherichia coli S5 that produces spectinomycin-resistance and cold sensitivity. This mutation significantly alters cell growth, folding of 16S ribosomal RNA, and translational fidelity. While translation initiation is not affected, both +1 and À1 frameshifting and nonsense suppression are greatly enhanced in the mutant strain. Interestingly, this S5 ribosome ambiguity-like mutation is spatially remote from previously identified S5 ribosome ambiguity (ram) mutations. This suggests that the mechanism responsible for ram phenotypes in the novel mutant strain is possibly distinct from those proposed for other known S5 (and S4) ram mutants. This study highlights the importance of S5 in ribosome function and cell physiology, and suggests that translational fidelity can be regulated in multiple ways.

It is known that an RNA’s structure determines its biological function, yet current RNA structure probing methods only capture partial structure information. The ability to measure intact (i.e., full length) RNA structures will facilitate investigations of the functions and regulation mechanisms of small RNAs and identify short fragments of functional sites. Here, we present icSHAPE-MaP, an approach combining in vivo selective 2′-hydroxyl acylation and mutational profiling to probe intact RNA structures. We further showcase the RNA structural landscape of substrates bound by human Dicer based on the combination of RNA immunoprecipitation pull-down and icSHAPE-MaP small RNA structural profiling. We discover distinct structural categories of Dicer substrates in correlation to both their binding affinity and cleavage efficiency. And by tertiary structural modeling constrained by icSHAPE-MaP RNA structural data, we find the spatial distance measuring as an influential parameter for Dicer cleavage-site selection.

SummaryA specific mutation of Escherichia coli ribosomal protein S5, in which glycine is changed to aspartate at position 28 [S5(G28D)], results in cold sensitivity and defects in ribosome biogenesis and translational fidelity. In an attempt to understand the roles of S5 in these essential cellular functions, we selected extragenic suppressors and identified rimJ as a high-copy suppressor of the cold-sensitive phenotype associated with the S5(G28D) mutation. Our studies indicate that RimJ overexpression suppresses the growth defects, anomalous ribosome profiles and mRNA misreading exhibited by the S5(G28D) mutant strain. Although previously characterized as the N-acetyltransferase of S5, our data indicate that RimJ, when devoid of acetyltransferase activity, can suppress S5(G28D) defects thus indicating that the suppression activity of RimJ is not dependent on its acetyltransferase activity. Additionally, RimJ appears to associate with pre-30S subunits indicating that it acts on the ribonucleoprotein particle. These findings suggest that RimJ has evolved dual functionality; it functions in r-protein acetylation and as a ribosome assembly factor in E. coli.