Long non-coding (lnc) RNAs (lect 5)

Question 1

What does it mean that the genome is transcribed pervasively?

Answer 1

almost all of the genome is transcribed
-even though a lot of it is non-coding (egs. siRNAs, rRNAs, tRNAs, etc)

Question 2

Types of non-coding RNAs

Answer 2

-lncRNA (long non-coding)
-siRNA (short interfering)
-miRNA (micro)
-snoRNA (short nucleolar)
-rRNA (ribosomal)
-tRNA (transfer)

Question 3

Why are classical RNAseq methods unsuitable for studying lncRNAs?

Answer 3

classical RNAseq uses polyA selection or d(T)priming
-only sequences processed RNA transcripts
-some lncRNAs are not processed

Instead: use nascent RNAseq methods eg. GROseq, PROseq, TTseq, NETseq

Question 4

Processing of lncRNAs

Answer 4

-5’ capping like for mRNA
-some lncRNAs spliced, others not
-some lncRNAs 5’ polyadenylated, others (eg. eRNAs, auRNAs) not

Question 5

Why are knockdown approaches unsuitable for testing lncRNA functions?

Answer 5

-deletions of lncRNAs may delete other DNA-encoded features
-lncRNAs not inactivated by frameshift muts or premature stop codons
-lncRNAs localised in nucleus not targeted by RNAi

Question 6

How are lncRNA functions tested?

Answer 6

-knockdown approaches don’t work well!
Instead:
-depleting lncRNA levels using antisense oligonuleotides or locked nucleic acids which are targeted and cleaved by RNase H
-CRISPR-Cas13 targeting

Question 7

What are cis-acting lncRNAs?

Answer 7

lncRNAs which affect the expression of genes near the locus of the lncRNA on the same chromosome

Question 8

What are trans-acting lncRNAs?

Answer 8

lncRNAs which affect the expression of genes on domains far from or on other chromosomes to the lncRNA locus
-act as signals, scaffolds (eg. HOTAIR) or guides
-tend to be more stable lncRNAs

Question 9

What characteristics make lncRNAs good transcriptional regulators?

Answer 9

-expression is tightly regulated
-have a short half life (are rapidly turned over)
-diverse structures allow lots of interactions with proteins (important for functions!)

Question 10

How can lncRNAs regulate transcription?

Answer 10

-recruit regulatory proteins (eg. multiple lncRNA interactions of PCR2 allows RNA-binding proteins and accessory proteins to be recruited)
-inhibit tf binding (eg. allosterically, directly or as decoys)
-altering chromatin, genome and nuclear structure (eg. eu and hetero- chromatin)
-being transcribed itself (regulates transcription of nearby genes)
-acting as scaffolds (eg. HOTAIR links functions of histone methyltransferase and histone demethylase)

Question 11

Canonical RNA-binding domains in proteins

Answer 11

seq-specific RNA binding
eg. RNA recognition motifs (RRM), zinc finger domains

Question 12

Non-canonical RNA-binding domains in proteins

Answer 12

more +ve-ly charged and disordered than canonical domains
lots present in proteins involved in gene regulation and chromatin organisation

Question 13

How do RNAs exist as conformational ensembles?

Answer 13

Can adopt multiple conformations
-conformation with lowest free energy is most highly populated
-based on conditions and mutations, conformation can change

Question 14

Xist

Answer 14

lncRNA which determines which X chromosome remains active and which is silenced
-chromatin remodelling

Question 14

MALAT-1

Answer 14

lncRNA localised in nucleus
-infrequently spliced
-uses alternative splicing and nuclear reorganisations to regulate expression of genes involved in spreading cancer

Question 14

Neat-1

Answer 14

lncRNA localised to nuclear paraspeckles which controls paraspeckle formation and structure
-overexpressed in cancer cells

Question 15

HOTAIR

Answer 15

lncRNA involved in gene regulation and chromatin remodelling in cancer