Regulation of gene expression by small RNAs Garrett A. Soukup Creighton University School of Medicine Department of Biomedical Sciences

Objectives • Appreciate that there are two related biochemical pathways through which small RNAs can affect gene expression • Understand how each pathway through its small RNA product (siRNA or miRNA) differently affects gene expression • Distinguish differences in biogenesis and action of siRNAs and miRNAs • Appreciate the biological roles and significance of siRNAs and miRNAs • Comprehend how small RNAs might be used as agents for biotechnological or therapeutic manipulation of gene expression

A tale of two pathways • RNA interference (RNAi) pathway: produces small interfering RNAs (siRNAs) that silence complementary target genes • MicroRNA pathway: produces microRNAs (miRNAs) that silence complementary target genes • Mechanisms involve transcriptional gene silencing (TGS) and/or post-transcriptional gene silencing (PTGS) • Pathways are conserved among most all eukaryotic organisms (fungi, protozoans, plants, nematodes, invertebrates, mammals)

RNAi pathway • 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 the siRNA strand

A brief history of RNAi • RNAi was initially discovered and characterized in the nematode worm, C. elegans • It was observed that double-stranded RNA (dsRNA) was 10-times more effectiv in silencing target gene expression than antisense or sense RNA alone • Genetic studies in C. elegans identified that the effect requires two components: Dicer and Argonaute • Andrew Fire (Stanford) and Craig Mellow (U Mass) were awarded the 2006 Nobel Prize in Medicine for their discovery of RNAi

Core components of the RNAi pathway • Dicer Dicer family proteins contain an N-terminal helicase domain, a C-terminal segment containing dual RNase III domains, and one or more dsRNA-binding motifs. Family members also contain a PAZ domain. • Argonaute (RISC complex) Argonaute family members are highly basic, ~100 kD proteins that contain PAZ and PIWI domains.

Utility of RNAi for functional genomics • siRNAs are powerful tools for manipulating gene expression and determining gene function

Synthetic siRNAs • Synthetic siRNAs that target any sequence can be prepared by chemical synthesis • In mammalian cells, siRNAs range in effectiveness at knocking down target gene expression (50-95%) • The effectiveness of an siRNA is dependent upon target sequence 5´-NNNNNNNNNNNNNNNNNNNUU-3´ ||||||||||||||||||| 3´-UUNNNNNNNNNNNNNNNNNNN-5´ sense antisense

Example of siRNA knockdown • siRNA targeting rev mRNA sequence encoding rev-EGFP fusion protein • Sense (S) or antisense (AS) strand of siRNA alone does not effect knockdown of rev-EGFP expression • An irrelevant siRNA sequence (IR) does not effect knockdown of rev-EGFP expression

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

Biological roles of RNAi • Cellular immune response to viruses (some organisms) • Genetic stability

Immune response • In certain organisms (especially plants), RNAi serves as a first line of defense against viral infection, as virus may contain or viral replication can produce dsRNA • To this point, a number of plant viruses encode proteins that specifically bind and sequester siRNAs as a means of countering the cellular immune response of RNAi

Genetic stability • RNAi represses transposable genetic elements in C. elegans and S. pombe • Disruption of Dicer or Argonaute increases the relative abundance of transposon RNA and increases transposon mobility • RNAi is required to establish and maintain heterochromatin formation and gene silencing at mating type loci and centromeres in S. pombe • Disruption of Dicer or Argonaute eliminates silencing, decreases histone and DNA methylation, and causes aberrant chromosome segregation • Highly repetitive DNA is often associated with heterochromatin which is transcriptionally silent

Mechanism effecting heterochromatin?

miRNA pathway • miRNAs are the products of endogenous genes • miRNAs function to post-transcriptionally repress target genes by inhibiting translation and/or decreasing mRNA half-life • One miRNA may effect many (e.g. hundreds) of target genes

A brief history of miRNAs • C. elegans was discovered to possess small noncoding RNAs (lin-4 and let-7) required to repress expression of target genes (lin-28 and lin-41) that direct developmental progress • At that time, these so-called small temporal RNAs (stRNAs) were found to repress translational of the target mRNAs by interacting with complementary sites in their 3’ untranslated regions (UTRs) • It was later appreciated that the stRNAs are processed by Dicer require Argonaute, and thus function through an RNAi-related pathway • With the subsequent discovery that there are many such small RNAs throughout eukaryotic organisms, the entire class was renamed microRNAs

Small but plenty • To date, nearly 8600 miRNA genes have been identified among 73 eukaryotic organisms (plants and animals) and 15 viruses • There are, for example 132 C. elegans, 78 Drosophila, 377 mouse, and 474 human miRNA genes • Approximately one third of miRNA genes are intronic with respect to protein coding genes • Approximately two thirds of miRNA genes are intergenic (independent genes) • Many miRNA genes are conserved among species

Conservation of miRNA sequence and structure • Certain miRNAs are highly conserved and thus evolutionarily ancient (e.g. let-7) • Sequence conservation must fulfill the require to form a dsRNA hairpin from which the miRNA is processed by Dicer

miRNA gene transcription • Most miRNA genes are transcribed by RNA Pol II • miRNA genes can be arrayed and thus co-expressed

The machinery: PAZ domains bind 3´ends

The machinery: Dicer recognition and cleavage of RNA

The machinery: Argonaute RNA binding and function

The machinery: Accessory factors

Argonaute proteins • Mammals possess 4 argonaute proteins (Ago1, Ago2, Ago3, and Ago4) • Only Ago2 has been demonstrated to possess RNA cleavage or “Slicer” activity • What, if any, are the distinctive roles of other Ago proteins?

Potential mechanisms

miRNA-target interaction • miRNA binding sites reside within the 3´ UTRs of target transcripts • Seed-pairing hypothesis (animal miRNAs) (miRNA nucleotides 2-7 and sometimes 8) • An aside: plant miRNAs differ in that they are entirely complementary to their target genes 5’ NNNNNNNA 3´ || ||| ||||||| 3´-NNNNNNNNNNNNNNNNNNNNN-5´ mRNA miRNA seed

Target gene identification • 3´ UTRs are typically highly divergent (not conserved) among otherwise highly conserved genes • Rationale: If miRNAs are conserved among species, so too should be their binding sites among conserved target genes • Based on the seed pairing hypothesis, bioinformatic algorithms search for conserved miRNA binding sites among conserved target genes • Due to the minimal base-pairing requirement, predicted target genes are numerous • Therefore, elucidating miRNA functions based on predicted target genes is difficult

miRNAs in development • miRNAs play various roles in cell proliferation, differentiation, fate determination, and differentiated cell function • miRNAs appear to contribute to transitions from stem (precursor) cells to differentiated cell types by refining/reinforcing desired gene expression profiles • miRNAs appear to “sharpen” developmental outcomes with regard to organogenesis, morphogenesis, and histogenesis

Differential expression of miRNAs among cell types: clues to function • Different cells express different miRNAs (e.g. stem cells versus differentiated cells) • miRNA expression is typically examined by microarray analysis or cloning and sequencing • miRNA expression domains within an organism are revealed by in situ hybridization (Locked Nucleic Acid probes) miR-1 miR-100 miR-375

Dicer knockout organisms • Knockout of Dicer disrupt RNAi and miRNA pathways • Conditional knockout of Dicer enables analysis of RNAi effects in specific tissues • Dicer knockout is embryonic lethal in mice Knockout embryos exhibit lack of stem cells, and cell proliferation is decreased • Conditional Dicer knockout mice display defects in morphohistogenesis Dicer knockout in certain tissues results in developmental delays, cell death, and aberrant gene expression

miRNAs in disease • Cancer cells exhibit distinct miRNA expression profiles • Aberrant miRNA expression can contribute to carcinogenesis

miRNAs as tumor suppressors and oncogenes