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

2. 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

3. 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)

4. 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

5. 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

6. 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.

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

8. 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´
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3´-UUNNNNNNNNNNNNNNNNNNN-5´
sense
antisense

9. 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

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

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

12. 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

13. 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

14. Mechanism effecting heterochromatin?

15. 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

16. 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

17. 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

18. 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

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

20. The machinery: PAZ domains bind 3´ends

21. The machinery: Dicer recognition and cleavage of RNA

22. The machinery: Argonaute RNA binding and function

23. The machinery: Accessory factors

24. 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?

25. Potential mechanisms

26. 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 ´
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3´-NNNNNNNNNNNNNNNNNNNNN-5´
mRNA
miRNA
seed

27. 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

28. 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

29. 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

30. 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

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

32. miRNAs as tumor suppressors and oncogenes