VL 28 (Isabel Bäurle)

Question 1

RNAi - How does it work?

Answer 1

RNAi:
gene expression silencing due to action of short RNA molecules

How does RNAi work?
1. required: long dsRNA
2. dsRNA processed by DICER → siRNA (20-25 nt)
3. siRNA associate with ARGONAUTE effector complex (RISC)
4. complementary mRNA (sequence specificity!) bound by RISC and inhibited

Where does dsRNA originate?
* Transgene (hairpin constructs)
* Complex transposon integration
* natural cis-antisense transcripts
* RNA-dependent RNA Polymerase (RDR)

Question 2

RISC Complex: ARGONAUTE Proteins bind siRNAs and inhibit their target mRNAs

Answer 2

• ARGONAUTE proteins cleave mRNA, inhibit translation, mediate transcriptional silencing
• Arabidopsis: 10 ARGONAUTE genes

Argonaute
* responsible for RNAi
* binds different small non-coding RNA classes
* small RNAs guide Argonaute to targets through base pairing→mRNA
cleavage, translation inhibition, mRNA decay initiation

Question 3

Diffrent Types of RNA interference

Answer 3

PTGS:
* viral transcription
→ dsRNA formation
→ diced by DCL2,4
→ siRNA
→ siRNA incorporated into AGO1
→AGO1associateswithotherproteins
→ RNA-induced silencing complex (RISC) formation
→ cleavage of complementary mRNA

TGS:
* Nuclear RNA Pol IV (Pol IV) generates ssRNA
* RDR2 converts transcripts → dsRNA + adds untemplated 3 ́ termina nucleotide to 2nd strands
* DCL3 cuts→24, 23 nt RNAs
→ siRNA incorporated into AGO4
→ siRNA-AGO4 complex finds target sites by binding to Pol V transcripts + interaction with CTD of Pol V largest SU
→ cytosine methyltransferase DRM2 recruited to Pol V-transcribed loci
→ RISC
→ de novo cytosine methylation

miRNA:
* MIR genes transcribed by PolII→pri-miRNAs→back-folding→hairpin
structure
* processing → pre-miRNAs
→DCL1
→ miRNA/miRNA+ duplexes
→ methylated by HEN1
→ transported to cytoplasm by HST1
→ miRNA incorporated into AGO1-containing RISC
→ mRNA cleavage/translation inhibition

Question 4

RNAi in C. elegans is induced by dsRNA:

Answer 4

• dsRNA injection → siRNA → gene inactivation trough degradation of endogenous mRNA
• Result: gene-specific loss-of-function/hypomorphic phenotypes; post- transcriptional + transcriptional gene silencing
• RNAi in C. elegans: systemic, heritable

Craig Mello & Andrew Fire (2006 NP)

dsRNA is the active Form

Question 5

Most siRNAs are produced from transposons and repetitive DNA:

Answer 5

• most cellular siRNAs derived from transposons, other repetitive sequences
• in Arabidopsis (seen in picture): high repeat density in pericentromeric chromosome regions

Question 6

Transposons

Answer 6

• can cause inactive or unstable alleles
• are abundant

Transposon silencing:
* many active transposons in maize
* mutants that interfere with epigenetic silencing → release transposons from silencing
* epigenetic marks though to have evolved to silence foreign DNA (transposons, viruses)

Question 7

siRNAs recruit DNA methylases and histone modifying enzymes to targets:

Answer 7

Question 8

MIR genes are transcribed into long
RNAs that are processed to miRNAs

Answer 8

• miRNAs encoded by MIR genes
• primary miRNA (pri-miRNA) transcript folds back into ds-structure → processed by DCL1
• miRNA+ strand is degraded

microRNAs - miRNAS
* thought to have evolved from siRNAs; produced, processed similarly
* encoded by MIR genes but act on other genes (= trans-acting regulatory factors)
* in plants: regulate developmentan, physiological events
* plants with small number of conserved miRNAs, large number on non-conserved miRNAs

Question 9

Conclusion

Answer 9

• Small RNAs contribute to the regulation and defense of the genome, and confer silencing specificity through base-pairing
• siRNA targets include repetitive-rich heterochromatin, transposons, viruses or other pathogens
• miRNAs and tasiRNAs targets include regulatory
  genes affecting developmental timing or patterning, nutrient homeostasis and stress responses