RNA-ligand interactions and control of gene expression

Outline • RNA interference (microRNAs and siRNAs) • Ribosomal RNA, tRNA, and mRNA interactions • RNA interactions affecting translation and transcription (outside the ribosome) • Naked RNA • RNA-metabolite interactions (riboswitches)

RNA interference

RNA interference (RNAi) • RNAi is the process whereby double-stranded RNA (dsRNA) induces homology-dependent degradation of cognate RNA (i.e. gene silencing) • RNAi is central to cellular mechanisms of post-transcriptional gene silencing (PTGS) and can also effect transcriptional gene silencing (TGS) • RNAi is highly conserved among eukaryotes (fungi, protozoans, plants, nematodes, invertebrates, mammals)

Overview of RNAi • Double-stranded RNA (dsRNA) is processed by Dicer, an RNase III family member, to produce 21-23nt small interfering RNAs (siRNAs) • siRNAs are manipulated by a multi-component nuclease called the RNA-induced silencing complex (RISC). • RISC specifically cleaves mRNAs that have perfect complementarity to an siRNA strand

Components of the RNAi pathway • Dicer Dicer belongs to the RNase III family of dsRNA-specific ribonucleases. Contains dsRNA-binding motifs and a PAZ domain believed to mediate protein-protein interactions. • Argonaute homologs (Dicer/RISC associated) Argonaute family members are highly basic proteins that contain PAZ and PIWI domains. Argonaute is the only identified component of RISC. • RNA-dependent RNA polymerase (RdRP) Endogenous RdRPs influence RNAi in certain eukaryotes including fungi, plants, C. elegans. Function to amplify the dsRNA signal.

Discovery of RNAi • RNAi was initially discovered and characterized in the C. elegans • It was observed that antisense or sense RNA could equally effect silencing (knock-down) of target gene expression • A later study demonstrated that antisense and sense RNA combined (dsRNA) is 10-times more effective in silencing target gene expression • Genetic studies in C. elegans have helped to identify genes encoding RNAi machinery

The bottom line • Any means by which siRNAs can be generated and delivered to a cell can elicit RNAi and gene-specific silencing • siRNAs are powerful tools for manipulating gene expression and determining gene function

Nature did not exhaust billions of years of evolution on RNAi solely for the benefit of modern day biologists!

Biological significance of RNAi • Cellular immune response to viruses In certain organism (especially plants), RNAi serves as a first line of defense against viral infection, as viral replication typically includes dsRNA species • Genetic stability RNAi repress the mobility of transposable genetic elements in C. elegans and S. pombe which requires the formation of dsRNA Revealed that RNAi can also effect transcriptional gene silencing by promoting heterochromatin formation (histone and DNA methylation) • Organismal development and germline fate Developmental processes are affected by endogenous non-coding RNAs that function through the RNAi pathway (microRNAs)

MicroRNAs (miRNAs) • miRNAs are products of endogenous genes and function through the RNA interference (RNAi) pathway to post-transcriptionally regulate the expression of other genes.

miRNA genes • To date, 1185 miRNA genes have been identified in various organisms, including approximately 200 in each human, mouse, and rat genomes. • Certain miRNAs are highly conserved among eukaryotic species.

miRNAs in development • miRNAs are differentially expressed among various tissue types and at various stages in cellular differentiation. e.g. there exist stem cell and neuronal cell specific miRNAs. • Disruption of RNAi pathway results in early embryonic lethality due to depletion of stem cells. • Certain miRNAs are known to control developmental timing and fate specification in C. elegans, leaf morphogenesis in plants, and hematopoetic lineage differentiation in mice.