small RNAs

Question 1

miRNAs

Answer 1

microRNAs

• 20-24 nt
• produced from larger precursor transcript (70-100) nt that have a hairpin structure
• hairpin cleaved by Dicer endonuclease enzyme to generate miRNAs
• located in various locations: intergenic, introns
• hundreds identified in eukaryotes – Illumina sequencing
• plant completely complementary
• animals not completely complementary

Question 2

miRNA biogenesis

Answer 2

initial transcript forms stem-loop structure (pri-miRNA)

• stem cleaved from rest of transcript (still have loop) (pre-miRNA)
• loop cleaved
• mature single stranded miRNA

Question 3

miRNA functions

Answer 3

1. post translational gene silencing/ transcript cleavage
   - incorporated into RISC complex where acts as guide to target and degrade Complementary mRNA by cleavage
   - down regulates gene expression
2. blocking translation
   can be involved in translational repression: form RISC that prevents binding of mRNA to ribosomes
   - down regulates gene expression (complete or partial)

Question 4

Draw miRNA mechanism

Answer 4

DRAW

Question 5

miRNA expression patterns

Answer 5

can be expressed in tissue specific manner and can be developmentally regulated

• miRNAs regulate many developmental processes, as well as other functions
• can have many targets or one target
• binding sites can have phenotypic effects (sheap muscle ex)

Question 6

families of small RNAs

Answer 6

classified into families based on sequences

• expand by gene duplication, contract by deletion
• some have early origins = roles in development
• recent origin= wider variety of roles
• some new miRNAs are derived from transposons and repeat sequences

Question 7

evolution of miRNAs in animals

Answer 7

• lineage specific miRNAs
• • many evolutionary recent miRNAs
• expression patterns in conserved miRNAs can vary
• • not strictly conserved even between 2 closely related species
• expression can change rapidly in evolutionary time

Question 8

acquiring a new miRNA

Answer 8

• initially expressed at low levels in specific spatio-temporal domain
• • many targets deleterious
• later most targets are neutral or advantageous and expressed at higher levels

Question 9

siRNAs

Answer 9

small interfering RNAS

• post transcriptional gene silencing
• no dedicated genes for siRNAs
• produced from double stranded RNAs
• some originate form TE and heterochromatin regions
• some help prevent expression of TEs
• FULLY complementary to targets

Question 10

siRNA mechanism

Answer 10

DRAW

- DICER, AGO, RISC

Question 11

siRNA silencing of retrotransposons

Answer 11

Dicer, Ago, risc binds TE mrRNA

- results in post transcriptional silencing of TE

Question 12

Inverted duplication and small RNAs

Answer 12

inverted duplication gene structure– mRNA forms loop structure
- major mode for how siRNA and miRNAs form de novo

Question 13

lncRNAs

Answer 13

long non-coding RNAs

• ~200nt and not translated into proteins
• many intergenic, some overlap with protein coding genes
• most recently identified
• some function in chromatin remodeling complexes to help regulate gene expression
• sequences evolve more rapidly than protein coding genes
• tissue specific
• less conservation

Question 14

Xist

Answer 14

lncRNA and x-chromosome inactivation

• expressed from inactive x chromosome
• 17kb= unusually large
• accumulates along x- chromosome to be inactivated and helps reduce heterochromatin formation

Question 15

functions of lncRNAs

Answer 15

• chromatin remodeling
• • recruitment of chromatin modifying complexes
• • decrease expression
• transcriptional control (recruit or block TF or bind promoter)
• • positive, negative, or negative by binding to promoter to form triplex (not common)
• reduce or inhibit function of miRNAs
• • bind miRNAs so fewer miRNAs available to reduce target gene
• – positive effect on gene regulation

Question 16

evolution of small RNAs

Answer 16

lncRNAs much less highly conserved than protein coding and less than small RNA precursors

• conservation = protein coding> small RNA> lncRNA
• lncRNA more recently evolved

Question 17

housekeeping vs regulatory ncRNAs

Answer 17

housekeeping
- constitutively expressed
- tRNA, rRNA, snRNA, snoRNA
regulatory
-lncRNA and small ncRNA
- 200 nt is cut off

Question 18

evolution of lncRNAs vs protein coding genes

Answer 18

lncRNA
- low sequence conservation
- maintain function by preserving short sequence stretches or structural motifs that serve as functional domains
protein coding
- high sequence conservation
- must preserve ORF to maintain function

Question 19

4 ways new lncRNAs can form

Answer 19

1. gradual transformation of protein coding to functional lncRNA
2. chromosome rearrangement of 2 un-transcribed regions
3. duplication of lncRNA to give rise to 2 lncRNA
4. TE insertion

Question 20

5S-OT

Answer 20

acts as a lncRNA
cis in mammals and trans in humans
- in mammals has cis and trans effects
- acts in cis to regulate its own expression
- acts in trans to modulate AS by interacting with the splice factor

Question 21

lncND

Answer 21

regulates itself and sequesters miRNA

- acts as a miRNA sponge

Question 22

3 ways miRNAs can originate

Answer 22

1. local or tandem duplication of existing miRNA followed by sub/neofunctionalization
2. gradual evolution of unstructured transcripts to form hairpin structures
3. antisense transcription of existing miRNA gene

Question 23

miRNA vs siRNA

Answer 23

same length

• mi= ENDOGENOUS
• si= exogenous and endogenous
• mi- don’t have to be fully complementary
• single stranded
• always have stem loop
• si - completely complementary
• single stranded
• no dedicated genes to them
• both use RISC complex
• doesn’t need to form a stem loop structure but can