Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing.

RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Here we show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation.     

A comprehensive view of regulation of gene expression by double-stranded RNA-mediated cell signaling

Double-stranded (ds) RNA, a common component of virus-infected cells, is a potent inducer of the type I interferon and other cellular genes. For identifying the full repertoire of human dsRNA-regulated genes, a cDNA microarray hybridization screening was conducted using mRNA from dsRNA-treated GRE cells. Because these cells lack all type I interferon genes, the possibility of gene induction by autocrine actions of interferon was eliminated. Our screen identified 175 dsRNA-stimulated genes (DSG) and 95 dsRNA-repressed genes. A subset of the DSGs was also induced by different inflammatory cytokines and viruses demonstrating interconnections among disparate signaling pathways. Functionally, the DSGs encode proteins involved in signaling, apoptosis, RNA synthesis, protein synthesis and processing, cell metabolism, transport, and structure. Induction of such a diverse family of genes by dsRNA has major implications in host-virus interactions and in the use of RNA(i) technology for functional ablation of specific genes.     

Control of endogenous gene expression timing by introns

Comparison of gene expression from transgenes and endogenous genes with or without introns reveals a time-regulating role of introns in natural biological systems.     

Growth hormone gene regulation: a paradigm for cell-type-specific gene activation

The growth hormone (GH) gene is specifically expressed within specialized cells of the anterior pituitary. The central role in GH gene activation is played by GHF-1, a homeodomain protein that is itself specifically expressed in the anterior pituitary.     

Coordinate regulation of small temporal RNAs at the onset of Drosophila metamorphosis

The lin-4 and let-7 small temporal RNAs play a central role in controlling the timing of Caenorhabditis elegans cell fate decisions. let-7 has been conserved through evolution, and its expression correlates with adult development in bilateral animals, including Drosophila [Nature 408 (2000), 86]. The best match for lin-4 in Drosophila, miR-125, is also expressed during pupal and adult stages of Drosophila development [Curr. Biol. 12 (2002), 735]. Here, we ask whether the steroid hormone ecdysone induces let-7 or miR-125 expression at the onset of metamorphosis, attempting to link a known temporal regulator in Drosophila with the heterochronic pathway defined in C. elegans. We find that let-7 and miR-125 are coordinately expressed in late larvae and prepupae, in synchrony with the high titer ecdysone pulses that initiate metamorphosis. Unexpectedly, however, their expression is neither dependent on the EcR ecdysone receptor nor inducible by ecdysone in cultured larval organs. Although let-7 and miR-125 can be induced by ecdysone in Kc tissue culture cells, their expression is significantly delayed relative to that seen in the animal. let-7 and miR-125 are encoded adjacent to one another in the genome, and their induction correlates with the transient appearance of an approximately 500-nt RNA transcribed from this region, providing a mechanism to explain their precise coordinate regulation. We conclude that a common precursor RNA containing both let-7 and miR-125 is induced independently of ecdysone in Drosophila, raising the possibility of a temporal signal that is distinct from the well-characterized ecdysone-EcR pathway.     

Epigenetic gene regulation by noncoding RNAs

Functional noncoding RNAs have distinct roles in epigenetic gene regulation. Large RNAs have been shown to control gene expression from a single locus (Tsix RNA), from chromosomal regions (Air RNA), and from entire chromosomes (roX and Xist RNAs). These RNAs regulate genes in cis; although the Drosophila roX RNAs can also function in trans. The chromatin modifications mediated by these RNAs can increase or decrease gene expression. These results suggest that the primary role of RNA molecules in epigenetic gene regulation is to restrict chromatin modifications to particular regions of the genome. However, given that RNA has been shown to be at the catalytic core of other ribonucleoprotein complexes, it is also possible that RNA also plays a role in modulating changes in chromatin structure.     

Effects of different target sites on antisense RNA-mediated regulation of gene expression

Antisense RNA is a type of noncoding RNA (ncRNA) that binds to complementary mRNA sequences and induces gene repression by inhibiting translation or degrading mRNA. Recently, several small ncRNAs (sRNAs) have been identified in Escherichia coli that act as antisense RNA mainly via base pairing with mRNA. The base pairing predominantly leads to gene repression, and in some cases, gene activation. In the current study, we examined how the location of target sites affects sRNA-mediated gene regulation. An efficient antisense RNA expression system was developed, and the effects of antisense RNAs on various target sites in a model mRNA were examined. The target sites of antisense RNAs suppressing gene expression were identified, not only in the translation initiation region (TIR) of mRNA, but also at the junction between the coding region and 3'' untranslated region. Surprisingly, an antisense RNA recognizing the upstream region of TIR enhanced gene expression through increasing mRNA stability. [BMB Reports 2014; 47(11): 619-624]     

RNA silencing: small RNAs as ubiquitous regulators of gene expression

'RNA silencing' is the suppression of gene expression through nucleotide sequence-specific interactions that are mediated by RNA. Initially identified as an immune system that is targeted against transposons and viruses, RNA silencing is emerging as a fundamental regulatory process that is likely to affect many layers of endogenous gene expression in most, if not all, eukaryotes.     

Characterization of MicA interactions suggests a potential novel means of gene regulation by small non-coding RNAs

MicA is a small non-coding RNA that regulates ompA mRNA translation in Escherichia coli. MicA has an inhibitory function, base pairing to the translation initiation region of target mRNAs through short sequences of complementarity, blocking their ribosome-binding sites. The MicA structure contains two stem loops, which impede its interaction with target mRNAs, and it is thought that the RNA chaperone protein Hfq, known to be involved in MicA regulation of ompA, may structurally remodel MicA to reveal the ompA-binding site for cognate pairing. To further characterize these interactions, we undertook biochemical and biophysical studies using native MicA and a ‘stabilized’ version, modified to mimic the conformational state of MicA where the ompA-binding site is exposed. Our data corroborate two proposed roles for Hfq: first, to bring both MicA and ompA into close proximity, and second, to restructure MicA to allow exposure of the ompA-binding site for pairing, thereby demonstrating the RNA chaperone function of Hfq. Additionally, at accumulated MicA levels, we identified a Mg²⁺-dependent self-association that occludes the ompA-recognition region. We discuss the potential contribution of an Mg²⁺-mediated conformational switch of MicA for the regulation of MicA function.