Emerging concepts of lncRNA function Flashcards
What is lncRNA?
Long non-coding RNA
What is pervasive transcription? (2)
- 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
What are examples of non-coding RNAs (ncRNAs)? (6)
- siRNA (small-interfering)
- rRNA (ribosomal)
- snoRNA (small nucleolar)
- miRNA (micro)
- tRNA (transfer)
- CUTs (cryptic upstream transcripts
How are lncRNAs defined? (5)
- 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
What processes are lncRNAs involved in? (7)
- mRNA stability
- mRNA translation
- mRNA sponge
- Histone modification
- Transcriptional regulation
- Splicing
- Chromatin interactions
What are example classifications of lncRNA? (4)
- PROMPT
- Gene body associated RNA (antisense/sense)
- lincRNA
(- eRNA)
What is a PROMPT? (3)
- Promoter upstream transcript
- Bidirectional transcription from start sites generates antisense promoter transcripts
- Roles include promoting looping between enhancers and promoters
What are gene body associated RNAs?
Produced from transcription initiation in introns in an antisense direction, can also be sense
What is lincRNA? (2)
- Long intergenic non-coding RNA
- Transcribed from non-coding regions between genes
What is eRNA? (2)
- Enhancer RNA
- Transcribed from enhancer regions and regulate gene expression
What are examples of lincRNAs? (2)
- MALAT-1 (Neat2)
- Neat1
What is the function of MALAT-1 (Neat2)?
Involved in alternative splicing and nuclear organisation
What is the function of Neat1?
Involved in paraspeckle formation
What are examples of lncRNAs? (3)
- Xist
- MALAT-1
- Neat1
What is the function of Xist?
X chromosome inactivation
What is a drawback of RNAseq? (2)
- RNAseq is based on polyA selection or oligo d(T) priming for cDNA production and sequencing
- Fails to detect unprocessed lncRNA transcripts
What is a better way of measuring transcription levels in a cell?
Mapping locations of transcribing RNA polymerases instead of RNAseq
What are examples of methods of nascent RNA transcription mapping? (4)
- GROseq
- PROseq
- NETseq
- TTseq
What is GROseq? (2)
- Global run on sequencing
- Bromouridine incorporated into RNA in vivo and used to purify newly synthesised RNAs
What is PROseq? (2)
- Precision nuclear run on sequencing
- Development of GRO-seq with all bromo-dNTPs which improves base-pair resolution
What is NETseq? (2)
- Negative elongating transcript sequencing
- Immunoprecipitate transcribing RNA polymerases, brings the RNA with it
What is TTseq? (2)
- Transient transcriptome sequencing
- Nascent RNA labelled with 4-thiouridine and purified
What is nascent RNA?
RNA that is being actively transcribed by so is still associated with RNA polymerase II
What do you see in nascent RNA sequencing that you don’t get from normal RNA seq?
Shows you transcription in all areas of the genome not just protein-coding/polyA enriched areas
How does 5’ and 3’ end processing differ in lncRNAs compared to mRNAs? (5)
- 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
What is the purpose of 3’ polyadenylation? (2)
- 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
How are lncRNAs structured? (4)
- 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)
How stable are lncRNA structures? (3)
- 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
How do you analyse RNA structures? (4)
- 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
Why are knockdown approaches for testing function challenging for lncRNAs? (4)
- 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)
What are examples of RNA structure probing techniques? (4)
- SHAPE (reagent modifies OH group)
- MaP (mutagenesis profiling)
- DMseq
- PARIS
What methods can be used to depleted lncRNA instead of normal knockdown? (2)
- Antisense oligonucleotides (ASO) e.g. locked nucleic acids (LNA)
- CRISPR-Cas13
What is ASO? (4)
- 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
What is CRISPR-Cas13?
RNA targeting CRISPR system
Why do lncRNAs make good transcriptional regulators? (4)
- 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
What is meant by cis-acting lncRNAs? (2)
- Affect expression of genes located proximal to the lncRNA locus on the same chromosome
- lncRNAs tend to act in cis
What is meant by trans-acting lncRNAs? (2)
- 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
What are examples of trans-acting lncRNAs? (5)
- HOTAIR
- Gas5
- lincRNAp21
- MALAT-1
- NEAT1
How do proteins bind to RNA? (2)
- 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)
What are canonical RNA binding motifs in proteins? (3)
- RNA recognition motif (RRM)
- hnRNP K-homology motif (KH)
- Zinc finger domains (ZnF)
How can lncRNAs regulate transcription? (4)
- 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
How do lncRNAs recruit regulatory protein complexes for transcription regulation? (4)
- Interact with nascent RNA during transcription
- Base-pairing with another RNA
- Base-pairing with DNA target sequence (R loop)
- Interact with another protein
How do lncRNAs inhibit binding of transcriptional regulators? (2)
- Act as a ‘decoy’
- Inhibit its activity by direct active site occlusion or allosteric modulation
How does transcription of lncRNAs regulate transcription?
Transcription of a lncRNA may regulate transcription of nearby mRNA genes
How can lncRNAs epigenetically regulate transcription?
Organising hetero or euchromatic regions and controlling posttranslational modifications of nearby chromatin
How are lncRNAs linked to polycomb proteins? (3)
- 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
What is the function of PRC2? (2)
- Polycomb protein
- Responsible for H3K27 methylation (silencing mechanism)
What is an example of a scaffolding function of lncRNA? (3)
- 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)
What are transcriptional condensates? (3)
- 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
