Programmed –1 ribosomal frameshifting (−1PRF) is a recoding mechanism to make alternative proteins from a single mRNA transcript. −1PRF is stimulated by cis-acting signals in mRNA, a seven-nucleotide slippery sequence and a downstream secondary structure element, which is often a pseudoknot. In this study we engineered the frameshifting pseudoknot from the mouse mammary tumor virus to respond to a rationally designed small molecule naphthyridine carbamate tetramer (NCTn). We demonstrate that NCTn can stabilize the pseudoknot structure in mRNA and activate –1PRF both in vitro and in human cells. The results illustrate how NCTn-inducible –1PRF may serve as an important component of the synthetic biology toolbox for the precise control of gene expression using small synthetic molecules.

G-Quadruplexes are noncanonical structures formed by guanine-rich regions of not only DNA but also RNA. RNA G-quadruplexes are widely present in the transcriptome as mRNAs and noncoding RNAs and take part in various essential functions in cells. Furthermore, stable RNA G-quadruplexes control the extent of biological functions, such as mRNA translation and antigen presentation. To understand and regulate the functions controlled by RNA G-quadruplexes in cellular environments, which are molecularly crowded, we would be required to investigate the stability of G-quadruplexes in molecular crowding. Here, we systematically investigated the thermodynamic stability of RNA G-quadruplexes with different numbers of G-quartets and lengths of loops. The molecular crowding conditions of polyethylene glycol with an average molecular weight of 200 (PEG200) were found to stabilize RNA G-quadruplexes with three and four G-quartets, while G-quadruplexes with two G-quartets did not exhibit any stabilization upon addition of PEG200. On the other hand, no difference in stabilization by PEG200 was observed among the G-quadruplexes with different loop lengths. Thermodynamic analysis of the RNA G-quadruplexes revealed more appropriate motifs for identifying G-quadruplex-forming sequences. The informatics analysis with new motifs demonstrated that the distributions of G-quadruplexes in human noncoding RNAs differed depending on the number of G-quartets. Therefore, RNA G-quadruplexes with different numbers of G-quartets may play different roles in response to environmental changes in cells.

No abstract

The mitogen-activated protein kinase (MAPK) cascade is a highly conserved signaling module composed of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKK) and MAPKs. The MAPKKK Mkh1 is an initiating kinase in Pmk1 MAPK signaling, which regulates cell integrity in fission yeast (Schizosaccharomyces pombe). Our genetic screen for regulators of Pmk1 signaling identified Shk1 kinase binding protein 5 (Skb5), an SH3-domain-containing adaptor protein.Here, we show that Skb5 serves as an inhibitor of Pmk1 MAPK signaling activation by downregulating Mkh1 localization to cell tips through its interaction with the SH3 domain. Consistent with this, the Mkh1 3PA mutant protein, with impaired Skb5 binding, remained in the cell tips, even when Skb5 was overproduced.

The hairpin structure is one of the most common secondary structures in RNA and holds a central position in the stream of RNA folding from a non-structured RNA to structurally complex and functional ribonucleoproteins. Since the RNA secondary structure is strongly correlated to the function and can be modulated by the binding of small molecules, we have investigated the modulation of RNA folding by a ligand-assisted formation of loop-loop complexes of two RNA hairpin loops. With a ligand (NCT6), designed based on the ligand binding to the G-G mismatches in double-stranded DNA, we successfully demonstrated the formation of both inter- and intra-molecular NCT6-assisted complex of two RNA hairpin loops. NCT6 selectively bound to the two hairpin loops containing (CGG)3 in the loop region. Native polyacrylamide gel electrophoresis analysis of two doubly-labeled RNA hairpin loops clearly showed the formation of intermolecular NCT6-assisted loop-loop complex. Förster resonance energy-transfer studies of RNA constructs containing two hairpin loops, in which each hairpin was labeled with Alexa488 and Cy3 fluorophores, showed the conformational change of the RNA constructs upon binding of NCT6. These experimental data showed that NCT6 simultaneously bound to two hairpin RNAs at the loop region, and can induce the conformational change of the RNA molecule. These data strongly support that NCT6 functions as molecular glue for two hairpin RNAs.