1. Steps in microRNA gene silencing
Transcription: Pol II transcribes miRNA gene, directs same modifications as an mRNA transcript
Steps in microRNA gene silencing
Pol II
5’Cap
polyA tail
Gene Silencing using different mechanisms

2. Steps in microRNA gene silencing
1. Transcription: Pol II transcribes miRNA gene, directs same modifications as an mRNA transcript
2. Cleavage: Drosha crops the miRNA transcript into an pre-miRNA
Pol II
5’Cap
polyA tail
Gene Silencing using different mechanisms

3. Steps in microRNA gene silencing
1. Transcription: Pol II transcribes miRNA gene, directs same modifications as an mRNA transcript
2. Cleavage: Drosha crops the miRNA transcript into an pre-miRNA
3. Nuclear export: gets pre-miRNA outside the nucleus
Pol II
5’Cap
polyA tail
Gene Silencing using different mechanisms

4. Steps in microRNA gene silencing
1. Transcription: Pol II transcribes miRNA gene, directs same modifications as an mRNA transcript
2. Cleavage: Drosha crops the miRNA transcript into an pre-miRNA
3. Nuclear export: gets pre-miRNA outside the nucleus
4. Dicing: Dicer cleaves pre-miRNA into ~22bp double-stranded RNA fragments
Pol II
5’Cap
polyA tail
Gene Silencing using different mechanisms

5. Steps in microRNA gene silencing
1. Transcription: Pol II transcribes miRNA gene, directs same modifications as an mRNA transcript
2. Cleavage: Drosha crops the miRNA transcript into an pre-miRNA
3. Nuclear export: gets pre-miRNA outside the nucleus
4. Dicing: Dicer cleaves pre-miRNA into ~22bp double-stranded RNA fragments
5. RISC: RNA-induced silencing complex. Binds to dsRNA , separates RNA strands and guides single stranded RNA to target
Pol II
5’Cap
polyA tail
Gene Silencing using different mechanisms

6. miRNAs tend to bind to the 3’UTR of a gene
In general,
microRNAs bind imperfectly to the mRNA target. This blocks translation.
Dicer
RISC
Translation is blocked!

7. miRNAs often show imperfect complementary
miRNA tends to bind in the 3’ UTR of target genes and are associated with the RISC complex
Not sure how this inhibits translation!
DICER: cleaves pre-mRNA in 22bp double-stranded RNA fragments
RISC: guides single-stranded RNA to the 3’UTR

8. miRNAs are prevalent genes
In animals, microRNAs are estimated to comprise around 1% of the predicted genes.
It is often difficult to discover microRNAs because they are small genes
Very difficult to identify the TARGETs of microRNAs as the complimentary binding is often imperfect.
The targets of miRNAs are very diverse

9. miRNA in Cancer You are studying the roles of miRNAs in cancer.
Oncogene: normally promotes cell growth
Tumor Suppressor: normally inhibits cell growth
Mutations in oncogenes result in up-regulation of the gene
Mutations in tumor suppressor genes result in down-regulation of the gene

10. miRNA in disease You are studying the roles of miRNAs in cancer.
How do miRNA genes affect transcription of their target genes?
How do miRNA genes affect translation of their target genes?
You identify a mutation in a miRNA gene in cancer patients. This miRNA normally inhibits the RAS oncogene.
Would you predict that these cancers patients have increased or decreased levels of RAS?
Would the mutation in the miRNA be considered a LOF or GOF?

11. LOF of miRNA = up-regulation of target gene
miRNA in disease
LOF of miRNA = up-regulation of target gene
A gene product inhibits growth
GOF miRNAs

12. miRNA in disease You are studying the roles of miRNAs in cancer.
You identify another mutation in a miRNA in cancer
patients; however, this miRNA normally inhibits the RB tumor suppressor.
Would you predict that these cancers patients have increased or decreased levels of RB tumor suppressor gene?
Would the mutation in the miRNA be considered a LOF or GOF?

13. GOF of miRNA = down-regulation of target gene
miRNA in disease
GOF of miRNA = down-regulation of target gene

14. miRNA in disease A gene product that promotes growth
A gene product inhibits growth

15. microRNA gene silencing and mRNA degradation

16. The RNA interference pathway
microRNAs bind imperfectly to their target sequences and inhibit translation mostly.
Double-stranded RNA molecules are used for RNA interference! Perfect complement = mRNA degradation

17. The RNA interference pathway
Naturally made, typically for viral protection
Dicer processing
RISC association
Synthetically made to knock down genes

18. Small interfering RNAs vs. MicroRNAs
Naturally made, typically for viral protection
Dicer processing
RISC association
Synthetically made to knock down genes
miRNAs tend to bind at the 3’UTR
siRNA bind anywhere on the mRNA

19. Small interfering RNAs vs. MicroRNAs
Small interferences RNAs
Example is synthetically produced double-stranded RNA
Binds with perfect complementary to its target sequence
Results in mRNA degradation
Uses a DICER and RISC complex
microRNAs
Examples are microRNA genes
Binds with imperfect complementary to its target sequence
Results in blockage of translation
Uses a DICER and RISC complex

20. The RNA interference pathway in Research
Think of a gene you want to knock down and use PCR to amplify it
Makes both sense and antisense transcripts = double-stranded RNA
Use your BIL333 skills to clone the gene into a special plasmid
Use your BIL333 skills to transform the plasmid into bacteria
Lisa Timmons & Andrew Fire
Nature 395, 854(29 October 1998)
doi: /27579
The worms eat the bacteria producing dsRNAs. RNAi is induced and gene is knocked-down! 

21. How do researchers produce dsRNA?
The T7 promoter flanks the gene of interest
If the T7 promoter is active, then two complimentary RNA molecules will be produced (sense and antisense)
The two complimentary RNA molecules will hydrogen bond forming dsRNA (double-stranded RNA).
The T7 promoter is recognized by a specific type of polymerase called the T7 RNA polymerase (this is viral polymerase).
Lisa Timmons & Andrew Fire
Nature 395, 854(29 October 1998)
doi: /27579
RNAi plasmid

22. How do researchers produce dsRNA?
The T7 RNA polymerase gene is found on the bacterial genomic DNA (not on the plasmid!)
The T7 RNA polymerase is not always expressed. It is under that control of the lac promoter. The lac promoter is only active in the presence of lactose (or a chemical analog known as IPTG).
When bacteria are subjected to IPTG, T7 polymerase is made.
T7 RNA polymerase will bind to the T7 sites on the plasmid and start producing dsRNA!
Lisa Timmons & Andrew Fire
Nature 395, 854(29 October 1998)
doi: /27579

23. Is RNAi reverse or forward genetics?
Think of a gene you want to knock down and use PCR to amplify it
Makes both sense and antisense transcripts = double-stranded RNA
Use your BIL333 skills to clone the gene into a special plasmid
Use your BIL333 skills to transform the plasmid into bacteria
Reverse
The worms eat the bacteria producing dsRNAs. RNAi is induced and gene is knocked-down!
REVERSE

24. The RNA interference pathway: Used extensively in Research\
RNA interference can be used to disrupt gene function. This is an example of Reverse Genetics!

25. Discovery of RNAi
Used RNA molecules targeting a gene that when knockdown produces a twitching phenotype.
Which version of the RNA induced a knock-down phenotype?
Why?
Lisa Timmons & Andrew Fire
Nature 395, 854(29 October 1998)
doi: /27579
Sense RNA= same sequence as the mRNA product
Antisense RNA= complimentary sequence as the mRNA product

26. The RNA interference pathway: Used extensively in Research
Lisa Timmons & Andrew Fire
Nature 395, 854(29 October 1998)
doi: /27579

27. Break!!!

28. Long noncoding RNAs
What are long non-coding RNAs (lnc-coding RNAs)?

29. Long noncoding RNAs What are long non-coding RNAs (lnc-coding RNAs)?
Difficult to define- A noncoding transcript greater than 200 nucleotides.
Found by analyzing transcriptome. A significant portion of RNAs did not have open reading frames (ie. a start and stop codon—thus no predicted protein sequence).

30. Long noncoding RNAs
How much of the genome is transcribed to long non-coding RNAs?

31. Long noncoding RNAs
How much of the genome is transcribed to long non-coding RNAs?
In transcriptome studies, data has suggestion that most of our genome is transcribed!

32. Long noncoding RNAs
What is the RNA world hypothesis?

33. Long noncoding RNAs What is the RNA world hypothesis?
Suggests that early life was based on RNA not DNA. This is because RNA is known to have catalytic functions
DNA is a more stable molecule and therefore became the informational storage molecule
Functional RNAs became limited to roles in translation and splicing while the major “role” of RNA was to link DNA to protein information
Do RNAs have more a functional role than first appreciated?

34. Long noncoding RNAs
Known functional roles of lncRNAs

35. lncRNAs can affect chromatin remodeling

36. lncRNAs can affect transcription
Recruit repressors
Recruit activators
Form a “triplex” but intercalating directly with DNA

37. lncRNAs can affect splicing and translation
Effect Splicing
Block translation directly

38. Long noncoding RNAs Functions

39. Xist Complex on X-Chromosome
Best Studied example is Xist
17kb RNA molecule that is essential in X-chromosome inactivation
Females are XX and Males are XY
Do females produce genes from both X chromosomes?

40. Xist Complex on X-Chromosome
What is Xist doing?
Xist recruits chromatin remodeling factors like the polycomb repressive complex
This alters the modifications on the histone tails
When Xist binds to the X chromosomes the histone show repressive marks
Excessive levels of H3K27me3– this is mark of transcriptionally silenced chromatin

41. Xist Complex on X-Chromosome
What type of protein do you think the polycomb repressive complex is?
The polycomb repressive complex is a histone methyltransferase!
Excessive levels of H3K27me3– this is mark of transcriptionally silenced chromatin

42. Xist Complex on X-Chromosome X-chromosome inactive
Why does only one X-chromosome become inactivated? Why is the other X-protected from becoming heterochromatin?
X-chromosome active
Produces an antisense transcript of Xist called Tsix. Therefore Xist is degraded
X-chromosome inactive
Less antisense transcript of Xist (Tsix)

43. Xist Complex on X-Chromosome

44. Regulatory RNAs in prokaryotes
sRNAs: bacterial regulatory RNAs that function in trans to control translation of binding sites
sRNAs are produced by its own gene
Bind to the 5’UTR of genes
Can have both POSITIVE and NEGATIVE regulation!
POSITIVE
Lisa Timmons & Andrew Fire
Nature 395, 854(29 October 1998)
doi: /27579
NEGATIVE

45. Regulatory RNAs in prokaryotes
Riboswitch RNA
The 5’ UTR region acts in CIS to control gene expression!
Aptamer binds a small molecule and undergoes a conformation change that can inhibit transcription or translation.

46. CRISPR: Immunity in Prokaryotes
Bits of virus DNA called “spacers” are inserted in between repetitive sequences
RNA transcript forms hairpins that are recognized by Cas proteins
Other Cas proteins process RNAs, which can degrade the viral DNA if attacked again