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

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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

miRNAseed

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