Noncoding RNA

Question 0

Classes on non coding RNAs

Answer 0

tRNAs
rRNAs
Spliceosomal RNAs
snoRNAs
piRNAs
Endogenous siRNAs
Enhancer RNAs
miRNAs
lincRNAs

Question 1

DNA –>RNA–>Protein

Answer 1

DNA-->DNA = DNA replication
DNA-->RNA = transcription (snRNAS)
RNA-->DNA = reverse transcription 
RNA-->RNA = RNA replication 
RNA-->protein = translation 
Protein--> protein = prion propagation

Question 2

Transfer RNAs

Answer 2

Responsible for translating codon–anticodon information into specific amino acids

Question 3

How many tRNAs in human genome?

Answer 3

About 500

Question 4

What was the first discovered noncoding RNA?

Answer 4

Yeast alanine tRNA(1965)

Cloverleaf Crystal structure first determined by X-ray crystallography in 1975

Question 5

Ribosomal RNAs

Answer 5

Along with ribosomal proteins, makes up the molecular machine that carried out translation

2 RNAs make up more than half the mass of the eukaryotic ribosome

Large subunit catalyzes peptide bond formation

Question 6

Role of RNAs in splicing

Answer 6

Spliceosomal

Self splicing introns

Question 7

Spliceosomal RNA

Answer 7

Spliceosome made up of 5 smaller nuclear ribonuclear proteins (snRNPs =snRNA + protein)

RNAs are involved in sequence recognition and possibly catalysis

Question 8

Self splicing introns

Answer 8

The intron itself forms ribozyme that catalyzes splicing (Cech 89)

Question 9

Heterochronic mutants

Answer 9

Mutations that disrupted developmental timing in C.elegans

Two mutants : Lin-14 and Lin-4

Question 10

lin-4 and lin-14

Answer 10

Acts as negative regulator of Lin-14

Ambros: Lin-14 alleles are completely epistatic to lin-4 alleles.

Turns out

Lin-4 represses lin-14 by binding to multiple complimentary strands of 3’UTR of lin-14

Question 11

MicroRNAs

Answer 11

> 1000 miRNAs have been discovered in the human genome

Lin-4

Can be transcribed as pre-miRNA or derived from introns of pre-mRNAs

Processed by Drosha or Spliceosome to form characteristic hairpin structure

Exported to the cytoplasm

Pre-miRNAs are cleaved by Dicer–>~22nt dsRNA

miRNA is incorporated into RNA-induced silencing complex (RISC) —gene silencing

Question 12

Gene silencing

Answer 12

Can occur through translation inhibition or target mRNA degradation

Question 13

miRNA discovery

Answer 13

Shotgun sequencing of small RNA molecules

Stem loop prediction

Covariance of substitution patterns

Question 14

Ambros

Answer 14

Predicted stem loops from genome sequence sequence and cloned 15 miRNAS

Question 15

Tuschl

Answer 15

Sequenced small RNAs form worms, flies, and humans

Question 16

Bartel

Answer 16

Sequenced 330 small RNAs to identify 55 new miRNAs

Question 17

miRNA target prediction

Computational predictions

Answer 17

1) Strong complementary between mRNA 3’UTR and miRNA (over seed region)
2) evolutionary constraint of seed region
3) accessibly secondary structure at UTR target sites
4) expression profiles of mRNAs and miRNAs

Question 18

miRNA target prediction

Experimental predictions

Answer 18

Over expression and knock down experiments

HITS-CLIP and related techniques

NGS library construction tricks to clone mRNA degradation products

Question 19

Long non-coding RNAs

Answer 19

> 200 bp
Transcribed by PolII, have poly A tails
Often lowly expressed, cell type restricted
Often poorly conserved

Question 20

Tiling microarrays and ngs

Answer 20

Allowed interrogation of genome wide transcription patterns

Question 21

Discovery lncRNAs

Answer 21

Expression: tiling microarrays and NGS
Chromatin modification signatures
 -H3K4me3 at promoters
 -H3K36me3 through gene body
Evolutionary conservation 
Differential expression

Question 22

Does lncRNAs contain an open reading frame?

Answer 22

Difficult to interpret because short open reading frames are likely to frequently occur by chance

Question 23

Are lncRNAs translated ?

Answer 23

Difficult to asses but modern. Proteomics approaches may begin to shed some light
Ribosomal profiling experiments gave been difficult to interpret

Pattern of evolutionary conservation takes advantage of differences between synonymous and no summits substations

Question 24

XIST

Answer 24

LncRNA
Mediates X chromosome silencing in mammals

Recruits PRC2 to form large repressed H3k27me3 domains across most of the X

Question 25

HOTAIR

Answer 25

Is expressed from the HoxC cluster, represses HOxD transcription in trans by enhancing recruitment of PRC2

Question 26

H19

Answer 26

Imprinted gene, involved in cell proliferation and body weight control

Mechanism unknown

Question 27

NRON

Answer 27

Regulates nuclear export

Question 28

How do lncRNAs function?

Answer 28

Functional roles maybe as diverse as proteins

Evidence from eQTL studies suggest that they do not often regulate genes in trans