ncRNAs

Question 1

Give at least three classes of ncRNAs

Answer 1

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs) – post-transcriptional
gene silencing
* Long non-coding RNAs (lncRNAs) – direct epigenetic machinery and establish
different epigenetic states
* Piwi-interacting RNAs (piRNAs) – control transposable elements and direct DNA
methylation at transposable elements

Question 2

Describe the process of miRNA formation

Answer 2

1. Transcription of miRNA gene -> pri-miRNA (* pri = primary transcripts)
2. Initial processing by DROSHA and PASHA -> pre-miRNA
3. Export to the cytoplasm by Exportin 5
4. Further procesing by DICER removing loop region
5. Assembly with RISC and Ago
6. Removal of passenger strand -> miRISC (containing only guide strand)
7. Incomplete binding of miRISC to target mRNA 3’UTR resulting in repressed translation, cleavage and degradation

Question 3

Describe the structural differences between pri-miRNA, pre-miRNA and mature miRNA

Answer 3

dsRNA_stem-loop structure with flanking ssRNA ends. This flanking ends are bing removed in the primary processing step and are missing in pre-miRNA. The mature miRNA would contain only the dsStem though as they get functionlalized in RISC the “passenger” strand will be removed

Question 4

Describe the process of siRNA formation

Answer 4

1. dsRNA from exogenous source (e.g. virus) or transcribed from a genome locus
2. Dicer digests part of the starting dsRNA -> siRNA
3. Assembly with RISC Ago2
4. Digestion of passenger strand -> activated RISC
5. Complementary binding to target strand.

Question 5

Examples of lncRNAs

Answer 5

X chromosome inactivation
(Xist/Tsix)
- Genomic imprinting
(Airn, Kcnq10t1, H19, etc)
- Hox gene silencing
(HOTAIR and HOTTIP)
- FLC silencing in plants
(COLDAIR)

Question 6

What is a particularly crucial role of lncRNA in epigenetics?

Answer 6

lncRNAs can direct
epigenetic complexes to
DNA (cis and trans)
Length and sequence of
the lncRNA provides
site-specificity!

*Possible mechanisms:
- Guides (Xist in cis, Kcnq10t1, Airn, HOTAIR in trans)
- Decoys (Jpx, MALAT1, Tsix)
- Signal (Xite, Ftx)
- Enhancers (eRNAs, in cis)
- Reservoir (H19)

Question 7

Some characteristics of lncRNAs

Answer 7

> 200bp, usually spliced, capped and polyadenylated
* Human genome is estimated to contain 16.000 lncRNAs!
* Developmentally regulated (brains express a LOT of lncRNAs)
* Mostly constrained to nucleus
* lncRNAs have very complex secondary structures

Question 8

Function of HOTAIR lncRNA

Answer 8

guides PRC2 complex towards the HOXD locus for a H3K27me3 and the LSD1 complex for demethylation of H3K4me2(*as well as other proteins for H2AK119Ub) => repression of expression.

*HOTAIR is considered an oncogene as it can inhibit tumour suppressors

Question 9

Function of COLDAIR

Answer 9

Vernalization: the process by which prolonged exposure to cold temperatures promotes flowering

Its gene is located in the intron of FLC (crucial for flowering in Arabidopsis.

During cold periods COLDAIR is being expressed (how?? magic). It then recruits PRC2 and PHD -> inhibits the expression of FLC.