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BIS477 World of RNA > Emerging concepts of lncRNA function > Flashcards

Emerging concepts of lncRNA function Flashcards

1
Q

What is lncRNA?

A

Long non-coding RNA

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2
Q

What is pervasive transcription? (2)

A
  • Transcription outside of protein coding areas of the genome
  • ENCODE project found ~75% of the human genome is transcribed producing lots of non-coding RNAs
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3
Q

What are examples of non-coding RNAs (ncRNAs)? (6)

A
  • siRNA (small-interfering)
  • rRNA (ribosomal)
  • snoRNA (small nucleolar)
  • miRNA (micro)
  • tRNA (transfer)
  • CUTs (cryptic upstream transcripts
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4
Q

How are lncRNAs defined? (5)

A
  • Hard to classify
  • Long RNA transcripts that DO NOT encode proteins
  • Longer than 200nt
  • Typically lack ORFs > 100 codons
  • Typically don’t display conserved codon usage
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5
Q

What processes are lncRNAs involved in? (7)

A
  • mRNA stability
  • mRNA translation
  • mRNA sponge
  • Histone modification
  • Transcriptional regulation
  • Splicing
  • Chromatin interactions
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6
Q

What are example classifications of lncRNA? (4)

A
  • PROMPT
  • Gene body associated RNA (antisense/sense)
  • lincRNA
    (- eRNA)
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7
Q

What is a PROMPT? (3)

A
  • Promoter upstream transcript
  • Bidirectional transcription from start sites generates antisense promoter transcripts
  • Roles include promoting looping between enhancers and promoters
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8
Q

What are gene body associated RNAs?

A

Produced from transcription initiation in introns in an antisense direction, can also be sense

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9
Q

What is lincRNA? (2)

A
  • Long intergenic non-coding RNA
  • Transcribed from non-coding regions between genes
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10
Q

What is eRNA? (2)

A
  • Enhancer RNA
  • Transcribed from enhancer regions and regulate gene expression
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11
Q

What are examples of lincRNAs? (2)

A
  • MALAT-1 (Neat2)
  • Neat1
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12
Q

What is the function of MALAT-1 (Neat2)?

A

Involved in alternative splicing and nuclear organisation

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13
Q

What is the function of Neat1?

A

Involved in paraspeckle formation

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14
Q

What are examples of lncRNAs? (3)

A
  • Xist
  • MALAT-1
  • Neat1
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15
Q

What is the function of Xist?

A

X chromosome inactivation

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16
Q

What is a drawback of RNAseq? (2)

A
  • RNAseq is based on polyA selection or oligo d(T) priming for cDNA production and sequencing
  • Fails to detect unprocessed lncRNA transcripts
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17
Q

What is a better way of measuring transcription levels in a cell?

A

Mapping locations of transcribing RNA polymerases instead of RNAseq

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18
Q

What are examples of methods of nascent RNA transcription mapping? (4)

A
  • GROseq
  • PROseq
  • NETseq
  • TTseq
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19
Q

What is GROseq? (2)

A
  • Global run on sequencing
  • Bromouridine incorporated into RNA in vivo and used to purify newly synthesised RNAs
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20
Q

What is PROseq? (2)

A
  • Precision nuclear run on sequencing
  • Development of GRO-seq with all bromo-dNTPs which improves base-pair resolution
21
Q

What is NETseq? (2)

A
  • Negative elongating transcript sequencing
  • Immunoprecipitate transcribing RNA polymerases, brings the RNA with it
22
Q

What is TTseq? (2)

A
  • Transient transcriptome sequencing
  • Nascent RNA labelled with 4-thiouridine and purified
23
Q

What is nascent RNA?

A

RNA that is being actively transcribed by so is still associated with RNA polymerase II

24
Q

What do you see in nascent RNA sequencing that you don’t get from normal RNA seq?

A

Shows you transcription in all areas of the genome not just protein-coding/polyA enriched areas

25
Q

How does 5’ and 3’ end processing differ in lncRNAs compared to mRNAs? (5)

A
  • lncRNA 5’ ends have the normal m7g cap
  • Splicing and 3’ end processing is less well defined
  • Many lncRNAs are 3’ polyadenylated
  • Some lncRNAs are also spliced
  • Many eRNAs and upstream antisense RNAs (uaRNAs/PROMPTs) are monoexonic and non-polyadenylated
26
Q

What is the purpose of 3’ polyadenylation? (2)

A
  • Increases stability of RNA transcripts so not turned over as quickly
  • If not very stable the RNA can’t diffuse very far to perform roles in different areas of the cell
27
Q

How are lncRNAs structured? (4)

A
  • Highly structured which generates multiple potential RNA:protein interaction modes
  • E.g. ribozymes and aptamers
  • E.g. HOTAIR
  • Watson-Crick base pairing forms stem loops with unpaired loops/bubbles, means there is great variability in potential for structure formation (secondary and tertiary)
28
Q

How stable are lncRNA structures? (3)

A
  • RNAs exist as conformational ensembles meaning they are very dynamic
  • Conformation with the lowest free energy is the most populated
  • Different factors can alter the conformational balance
29
Q

How do you analyse RNA structures? (4)

A
  • Hard to crystallise because RNAs are sensitive to degradation
  • Chemical probes are modify areas of the RNA based on its availability to the solvent i.e. accessibility is lower in a base-paired region compared to single stranded region so can measure the base-pairing status
  • Modified nucleotides are then misincorporated during reverse transcription step of sequencing to make cDNA so detect mutation
  • High mutational frequency = single stranded
30
Q

Why are knockdown approaches for testing function challenging for lncRNAs? (4)

A
  • Deletions that remove a lncRNA gene may also alter other DNA-encoded features
  • lncRNAs are not inactivated by premature stop codons or frameshift mutations
  • lncRNAs often arise in functionally important regions and introns
  • Nuclear localised lncRNAs aren’t targeted by RNAi machinery (tends to be in cytoplasm)
30
Q

What are examples of RNA structure probing techniques? (4)

A
  • SHAPE (reagent modifies OH group)
  • MaP (mutagenesis profiling)
  • DMseq
  • PARIS
31
Q

What methods can be used to depleted lncRNA instead of normal knockdown? (2)

A
  • Antisense oligonucleotides (ASO) e.g. locked nucleic acids (LNA)
  • CRISPR-Cas13
32
Q

What is ASO? (4)

A
  • Antisense oligonucleotides
  • Short modified oligonucleotides complementary to target RNA sequence
  • RNase H mediated degradation of RNA/ASO or RNA/LNA hybrids
  • E.g. locked nucleic acids (LNA) are linked by a methylene bridge which provides optimal conformation for Watson-Crick base pairing and allows rapid and stable duplex formation
33
Q

What is CRISPR-Cas13?

A

RNA targeting CRISPR system

34
Q

Why do lncRNAs make good transcriptional regulators? (4)

A
  • Tightly controlled spatial and temporal expression (quite unstable)
  • Short half-life (rapid turnover)
  • Very big so can make multiple interactions with proteins through diverse RNA binding motifs
  • Protein:RNA interactions are critical for lncRNA function
35
Q

What is meant by cis-acting lncRNAs? (2)

A
  • Affect expression of genes located proximal to the lncRNA locus on the same chromosome
  • lncRNAs tend to act in cis
36
Q

What is meant by trans-acting lncRNAs? (2)

A
  • Act as signals, scaffolds or guides to affect expression of genes on distant domains on the same chromosome or on different chromosomes
  • Tends to favour more stable processed lncRNAs
37
Q

What are examples of trans-acting lncRNAs? (5)

A
  • HOTAIR
  • Gas5
  • lincRNAp21
  • MALAT-1
  • NEAT1
38
Q

How do proteins bind to RNA? (2)

A
  • Sequence-specific RNA binding is typically mediated by canonical RNA binding motifs in proteins
  • There are many non-canonical RNA binding domains which tend to be in proteins involved in chromatin organisation/gene regulation, unstructured and enriched for positive charge amino acids (arginine and lysine)
39
Q

What are canonical RNA binding motifs in proteins? (3)

A
  • RNA recognition motif (RRM)
  • hnRNP K-homology motif (KH)
  • Zinc finger domains (ZnF)
40
Q

How can lncRNAs regulate transcription? (4)

A
  • Recruitment of regulatory protein complexes
  • Inhibit binding of transcriptional regulators
  • Through the act of being transcribed
  • By altering epigenetic modifications of chromatin, genome organisation or nuclear architecture
41
Q

How do lncRNAs recruit regulatory protein complexes for transcription regulation? (4)

A
  • Interact with nascent RNA during transcription
  • Base-pairing with another RNA
  • Base-pairing with DNA target sequence (R loop)
  • Interact with another protein
42
Q

How do lncRNAs inhibit binding of transcriptional regulators? (2)

A
  • Act as a ‘decoy’
  • Inhibit its activity by direct active site occlusion or allosteric modulation
43
Q

How does transcription of lncRNAs regulate transcription?

A

Transcription of a lncRNA may regulate transcription of nearby mRNA genes

44
Q

How can lncRNAs epigenetically regulate transcription?

A

Organising hetero or euchromatic regions and controlling posttranslational modifications of nearby chromatin

45
Q

How are lncRNAs linked to polycomb proteins? (3)

A
  • PRC2 role in epigenetic transcriptional silencing during embryonic development and in cancer
  • Makes multiple lncRNA interactions
  • Has an RNA binding region in the core Ezh2 subunit
46
Q

What is the function of PRC2? (2)

A
  • Polycomb protein
  • Responsible for H3K27 methylation (silencing mechanism)
47
Q

What is an example of a scaffolding function of lncRNA? (3)

A
  • HOTAIR scaffolds an interaction between a histone methyltransferase and a histone demethylase
  • Linking 2 different and complementary modification processes in one area of the genome
  • Removal of H3K4 methylation by the demethylase (positive regulator of transcription) and subsequent addition of H3K27 methylation (negative regulator of transcription)
48
Q

What are transcriptional condensates? (3)

A
  • Regions of the nucleus with lots of transcription factors and RNA polymerase and are associated with increased transcription
  • lncRNAs can control which transcription factors are recruited into the condensate and its material properties
  • Formation of the condensate is dependent on the level/length of different RNA transcripts

BIS477 World of RNA (14 decks)

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