Long non-coding RNAs

Question 1

Which RNAs are canonically considered non-coding?

Answer 1

Any RNA which doesn’t get translated and has endogenous activity.

Examples:
1. tRNAs
2. rRNAs
3. snoRNAs
4. snRNAs
5. piRNAs

Question 2

Which RNAs are canonically considered long non-coding?

Answer 2

Any RNA which doesn’t get translated and has endogenous activity.

Examples:
1. tRNAs
2. rRNAs
3. snoRNAs
4. snRNAs
5. piRNAs

Question 3

What correlates with species complexity?

Answer 3

non-coding RNAs in the genome. These lead to lots of regulation of the ~2% genome.

Question 4

RNA can form non-canonical base pairs which lead to them being able to generate complex tertiary structures, including triplexes. Which non-canonical base pairs?

Answer 4

U : G
U : A

Question 5

There are >16k lncRNAs, which polymerases encode which RNAs?

Answer 5

RNApol1 : rRNA
RNApol2 : mRNA, snoRNA, lncRNA, sncRNA
RNApol3 : tRNA, snRNA

Question 6

What defines a long non-coding RNA?

Answer 6

1. > 200bp.
2. Downstream of RNApol2
3. Can cis- and/or transregulate chromatin

Question 7

There are two core proteins that lncRNAs interact with depending on if they cis- or transr-egulate the chromatin, which?

Answer 7

Cis : HOTTIP
Trans : HOTAIR

Question 8

How does X-chromosome inactivation work in females?

Answer 8

The X-chromosome inactivation center (XIC) transcribes XIST (X-inactive specific transcript) and the antisense TSIX which represses XIST, and vice versa. When there’s a surplus of XIST, it coats the chromosome from which it derives and recruits hyperacetylated histone proteins (MacroH2A, H3, H4) –> chromatin condensation –> inactivation.

HOTAR interacts with XIST on the chromosome which is to be inactivated. (??)

Question 9

What’s the function of HOTAIR?

Answer 9

HOTAIR = HOX transcript antisense RNA.

It represses the expression of the HOX-D cluster in a similar way that XIST represses its X-chromosome.

Question 10

Apart from the mechanisms underlying X-chromosome- and HOX inactivation, what processes are lncRNAs known for regulating?

Answer 10

1. Regulation of splicing via Alu/SINEB2, PTV1 and MALAT1
2. Production of microproteins (function unknown). The microproteins often have the opposite regulatory function as the corresponding lncRNA.
   (The mechanism may be the same as of HOTAIR-HOXD and XIST-TSIX.)
3. Acting like decoy RNA cytosolically, regulating chemical biological equilibriums and translations.
4. Can generate paraspeckles, irregular structures in the nucleus.

Question 11

What’s chIRP-MS? (comprehensive identification of RNA binding proteins)

Answer 11

It’s a method of precipitating and characterising RNA binding proteins.

1. Cross link cells in vivo.
2. Lyse cells, add biotinyolated oligos complementary to the target RNA.
3. Purify the lysate w/ streptavidin magnetic beads which bind the biotin.
4. Perform biotin elution.
5. Perform M/S.

Question 12

How are circular RNAs formed?

Answer 12

Circular lncRNAs are formed through back-splicing. This is when two splice sites become covalently bounded. 3’/5’ flanking complementary Alu sequences catalyze the formation of circRNAs (Alu is a repetitive element).

circRNAs can contain exons as well as introns.

circRNAs are more stable than linear RNA.

Question 13

What’s the functions of circular RNAs (4)?

Answer 13

1. miRNA sponges (miRNA hosts small seed sequences which are bound by the circRNA.
2. RBP sponges (contains binding sites)
3. Recruits chromatin modifiers
4. Template for translation.

Question 14

What’s ribosome profiling?

Answer 14

You find the RNA sites to which ribosomes bind. If you know where a protein binds, you can assay its responsiveness to stimuli this way.
1. Lyse cells.
2. Fixate RNA-protein interactions with paraffin.
3. Treat lysate extensively w/ lots of nucleases.
4. Sequence the product. Any shielded RNA will remain.