We have identified the S(MK) box as a conserved RNA motif in the 5' untranslated leader region of metK (SAM synthetase) genes in lactic acid bacteria, including Enterococcus, Streptococcus and Lactococcus species. This RNA element bound SAM in vitro, and binding of SAM caused an RNA structural rearrangement that resulted in sequestration of the Shine-Dalgarno (SD) sequence. Mutations that disrupted pairing between the SD region and a sequence complementary to the SD blocked SAM binding, whereas compensatory mutations that restored pairing restored SAM binding. The Enterococcus faecalis S(MK) box conferred translational repression of a lacZ reporter when cells were grown under conditions where SAM pools are elevated, and mutations that blocked SAM binding resulted in loss of repression, demonstrating that the S(MK) box is functional in vivo. The S(MK) box therefore represents a new SAM-binding riboswitch distinct from the previously identified S box RNAs.

Three distinct classes of S-adenosyl-l-methionine (SAM)-responsive riboswitches have been identified that regulate bacterial gene expression at the levels of transcription attenuation or translation inhibition. The SMK box (SAM-III) translational riboswitch has been identified in the SAM synthetase gene in members of the Lactobacillales. Here we report the 2.2-Å crystal structure of the Enterococcus faecalis SMK box riboswitch. The Y-shaped riboswitch organizes its conserved nucleotides around a three-way junction for SAM recognition. The Shine-Dalgarno sequence, which is sequestered by base-pairing with the anti–Shine-Dalgarno sequence in response to SAM binding, also directly participates in SAM recognition. The riboswitch makes extensive interactions with the adenosine and sulfonium moieties of SAM but does not appear to recognize the tail of the methionine moiety. We captured a structural snapshot of the SMK box riboswitch sampling the near-cognate ligand S-adenosyl-l-homocysteine (SAH) in which SAH was found to adopt an alternative conformation and fails to make several key interactions.

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RNAi is a conserved mechanism in which small interfering RNAs (siRNAs) guide the degradation of cognate RNAs, but also promote heterochromatin assembly at repetitive DNA elements such as centromeric repeats1,2. However, the full extent of RNAi functions and its endogenous targets have not been explored. Here we show that in the fission yeast Schizosaccharomyces pombe, RNAi and heterochromatin factors cooperate to silence diverse loci, including sexual differentiation genes, genes encoding transmembrane proteins, and retrotransposons that are also targeted by the exosome RNA degradation machinery. In the absence of the exosome, transcripts are processed preferentially by the RNAi machinery, revealing siRNA clusters and corresponding increase in heterochromatin modifications across large domains containing genes and retrotransposons. We show that the generation of siRNAs and heterochromatin assembly by RNAi is triggered by a mechanism involving the canonical poly(A) polymerase Pla1 and an associated RNA surveillance factor Red1, which also activate the exosome. Remarkably, siRNA production and heterochromatin modifications at these target loci are regulated by environmental growth conditions, and by developmental signals that induce gene expression during sexual differentiation. Our analyses uncover interplay between RNAi and the exosome that is conserved in higher eukaryotes, and show that differentiation signals modulate RNAi silencing to regulate developmental genes.

T4 RNA ligases are commonly used to attach adapters to RNAs, but large differences in ligation efficiency make detection and quantitation problematic. We developed a ligation selection strategy using random RNAs in combination with high-throughput sequencing to gain insight into the differences in efficiency of ligating pre-adenylated DNA adapters to RNA 3′-ends. After analyzing biases in RNA sequence, secondary structure and RNA-adapter cofold structure, we conclude that T4 RNA ligases do not show significant primary sequence preference in RNA substrates, but are biased against structural features within RNAs and adapters. Specifically, RNAs with less than three unstructured nucleotides at the 3′-end and RNAs that are predicted to cofold with an adapter in unfavorable structures are likely to be poorly ligated. The effect of RNA-adapter cofold structures on ligation is supported by experiments where the ligation efficiency of specific miRNAs was changed by designing adapters to alter cofold structure. In addition, we show that using adapters with randomized regions results in higher ligation efficiency and reduced ligation bias. We propose that using randomized adapters may improve RNA representation in experiments that include a 3′-adapter ligation step.