In bacteria, small non-coding RNAs (sRNAs) could function in gene regulations under variable stress responses. DsrA is an ∼90-nucleotide Hfq-dependent sRNA found in Escherichia coli. It regulates the translation and degradation of multiple mRNAs, such as rpoS, hns, mreB and rbsD mRNAs. However, its functional structure and particularly how it regulates multiple mRNAs remain obscure. Using NMR, we investigated the solution structures of the full-length and isolated stem–loops of DsrA. We first solved the NMR structure of the first stem–loop (SL1), and further studied the melting process of the SL1 induced by the base-pairing with the rpoS mRNA and the A-form duplex formation of the DsrA/rpoS complex. The secondary structure of the second stem–loop (SL2) was also determined, which contains a lower stem and an upper stem with distinctive stability. Interestingly, two conformational states of SL2 in dynamic equilibrium were observed in our NMR spectra, suggesting that the conformational selection may occur during the base-pairing between DsrA and mRNAs. In summary, our study suggests that the conformational plasticity of DsrA may represent a special mechanism sRNA employed to deal with its multiple regulatory targets of mRNA.

In Drosophila , dosage compensation globally upregulates the expression of genes located on male single X-chromosome. Maleless (MLE) helicase plays an essential role to incorporate the roX lncRNA into the dosage compensation complex (MSL-DCC), and such function is essentially dependent on its dsRNA-binding domains (dsRBDs). Here, we report a 2.90Å crystal structure of tandem dsRBDs of MLE in complex with a 55mer stem-loop of roX2 (R2H1). MLE dsRBDs bind to R2H1 cooperatively and interact with two successive minor grooves and a major groove of R2H1, respectively. The recognition of R2H1 by MLE dsRBDs involves both shape- and sequence-specificity. Moreover, dsRBD 2 displays a stronger RNA affinity than dsRBD 1 , and mutations of key residues in either MLE dsRBD remarkably reduce their affinities for roX2 both in vitro and in vivo . In Drosophila , the structure-based mle mutations generated using the CRISPR/Cas9 system, are partially male-lethal and indicate the inter-regulation among the components of the MSL-DCC at multiple levels. Hence, our research provides structural insights into the interactions between MLE dsRBDs and R2H1 and facilitates a deeper understanding of the mechanism by which MLE tandem dsRBDs play an indispensable role in specific recognition of roX and the assembly of the MSL-DCC in Drosophila dosage compensation.

MEX-3 is a K-homology (KH) domain-containing RNA-binding protein first identified as a translational repressor in , and its four orthologs (MEX-3A-D) in human and mouse were subsequently found to have E3 ubiquitin ligase activity mediated by a RING domain and critical for RNA degradation. Current evidence implicates human MEX-3C in many essential biological processes and suggests a strong connection with immune diseases and carcinogenesis. The highly conserved dual KH domains in MEX-3 proteins enable RNA binding and are essential for the recognition of the 3'-UTR and post-transcriptional regulation of MEX-3 target transcripts. However, the molecular mechanisms of translational repression and the consensus RNA sequence recognized by the MEX-3C KH domain are unknown. Here, using X-ray crystallography and isothermal titration calorimetry, we investigated the RNA-binding activity and selectivity of human MEX-3C dual KH domains. Our high-resolution crystal structures of individual KH domains complexed with a noncanonical U-rich and a GA-rich RNA sequence revealed that the KH1/2 domains of human MEX-3C bound MRE10, a 10-mer RNA (5'-CAGAGUUUAG-3') consisting of an eight-nucleotide MEX-3-recognition element (MRE) motif, with high affinity. Of note, we also identified a consensus RNA motif recognized by human MEX-3C. The potential RNA-binding sites in the 3'-UTR of the human leukocyte antigen serotype () mRNA were mapped with this RNA-binding motif and further confirmed by fluorescence polarization. The binding motif identified here will provide valuable information for future investigations of the functional pathways controlled by human MEX-3C and for predicting potential mRNAs regulated by this enzyme.

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