L30 Long non-coding RNA

Question 1

Features of long non-coding RNAs (lncRNAs)

Answer 1

see onenote

• regulate gene activity
• vast majority have no known function

Question 2

Proposed function of lncRNA

Answer 2

see onenote

Question 3

Function of lncRNAs - tethers

Answer 3

see onenote

• lncRNA remains tethered to the encoding locus during transcription
• protein binding motif at 5’ end recruits DNA/histone modifying enzymes
• modify chromatin of adjacent sites on the same chromosome, regulation occurs in cis

Question 4

Function of lncRNAs - guides

Answer 4

see onenote

• lncRNA released from site of transcription
• regulation in cis or trans

Question 5

Hox gene regulation example

Answer 5

see onenote

multiple lncRNA present in hox clusters

targeted knockout of hotair gene led to homeotic conversion of lumbar to sacral region

Question 6

Hotair regulates chromatin state

Answer 6

see onenote

hotair functions as a guide in trans
- promotes repressive chromatin state in trans

Question 7

Hotair - global regulator of gene expression

Answer 7

regulates many other genes, not sequence specific

ecotopic hotair detected in some human cancers - associated with metastasis/progression
- repressive chromatin is lost from transcriptional start sites of upregulated genes

Question 8

X chromosome inactivation

Answer 8

see onenote

Question 9

Female calico cats are mosaics

Answer 9

see onenote

x inactivation results in two populations of cells expressing different fur pigmentation - only in heterozygous females

Question 10

Stages of random XCI in mammals

Answer 10

see onenote

Once embryo becomes implanted in utero => two clonal populations that represent different x inactivated chromosomes

Question 11

XCI associated with heterochromatin formation

Answer 11

• X selected for silencing (Xi) is coated in lncRNA called Xist
• Xi condenses to form heterochromatin and recruits histone variants
• fully condensed Xi (Barr body) migrates to nuclear periphery
• majority of X-linked genes silenced but a few remain active

Question 12

X-inactivation centre (XIC)

Answer 12

see onenote

• <500kb region
• source of seven different lncRNA e.g. Xist, Tsix, Xite _ small ncRNAs
• > 7 lncRNA ensure orderly progression through XCI - all are involved in regulating Xist

Question 13

Xist - X inactive specific transcript

Answer 13

see onenote slides

• 17kb, spliced, polyA non-coding transcript
• constrained to nucleus
• initially expressed at low levels from both X chromosomes in undifferentiated embryonic cells

Question 14

Xist acts as cis guide

Answer 14

see onenote

Xis lncRNA has many repeat motifs

E.g.
Xist interacts with SPEN, binds to histone deactylase => removes histone activation mark from histone tails

Question 15

Xist triggers chromatin modification

Answer 15

• PCR2 recruited to Xi by unknown mechanism
• deposit H3K27me3 and other silencing marks
• extensive CG methylation in gene promoters

Question 16

Secondary chromatin modification

Answer 16

see onenote

Chromosome wide silencing not sufficient for x inactivation, needs further recruitment of chromatin modifying factors - secondary silencing factors that recognised the silenced chromosome, binds to it and causes further formation of heterochromatin

Question 17

lncRNA controls Xist activity

Answer 17

see onenote

• 40kb antisense Tsix transcript has motifs that are also bound by chromatin modifying factors (CMF) - acts as decoy to control Xist activity in cis

Question 18

Choosing the X chromosome to be activated

Answer 18

see onenote

• first event in XCI involves brief pairing between X chromosomes
• involves Tsix lncRNA physically interacting with protein to form a protein-RNA bridge

Question 19

X-X pairing and establishment of Xi

Answer 19

Paired X chromosomes exchange bound

• bound factors accumulate on just one X chromsome (future Xa)
• The one without the activation factors lose tsix expression and will become the inactivated x chromsome

Distribution of bound factors determines Tsix expression
- chromosome lacking bound factors (future Xi) - Tsix no longer expressed, increase in repressive H3k7me marks, Xist expression elevated