L19 - Non-coding RNAs and mechanisms controlling pervasive transcription in eukaryotic cells

What is the only mRNA which is not polyadenylated?

Histones

What % do coding regions in higher eukaryotes make up?

Coding regions in higher eukaryotes make up 1-2% of the total genome

What is non-coding RNA?

“The term non-coding RNA (ncRNA) is commonly employed for RNA that does not encode a protein, but this does not mean that such RNAs do not contain information nor have function”

Where are most ncRNAs located?

Many ncRNAs retain in the nucleus, they are exported to the cytoplasm, they are very quickly degraded or stabilised

Which ncRNAs are transcribed by RNA Polymerase II (Pol II)?

• small nuclear and small nucleolar RNAs (snRNA and snoRNAs)

• long intergenic RNAs (lincRNAs)

• enhancer RNAs (eRNAs)

• promoter-associated ncRNAs (PROMPTs, paRNAs, TSS-ncRNAs)

• many more short classes: miRNAs, piRNAs, telsRNAs, siRNAs

Which ncRNAs are long ncRNAs (lncRNAs)?

• Over 200 nts

• eg lincRNAs, eRNAs, PROMPTs

Which ncRNAs are transcribed by RNA Polymerase I and III?

• rRNA

• tRNA

How are ncRNAs separated?

In general, we separate ncRNAs into those which are dependently synthesised (co-expressed with mRNA) and independently synthesized (expressed from their own promoters)

What are the different types of ncRNAs associated with protein-coding genes (dependent ncRNAs)?

• ncRNAs originating from nucleosome free regions

• ncRNAs processed from pre-mRNA

What are ncRNAs originating from nucleosome free regions?

o promoter-associated RNAs

o Transcription from promoter of protein-coding gene results in mRNA and ncRNA

o Short ncRNA produced in the same direction as RNA (into the coding sequence)

What are the promoter associated ncRNA classes?

• paRNA/TSS-ncRNAs (promoter-associated RNA/ transcription start site ncRNA)

• PROMPTs (promoter upstream transcripts)

What are TSS-ncRNAs?

o transcribed in the same direction as the coding/sense sequence

o may make up to 80-90% of sense transcription

o 20– 60 nt long

o non-functional

o products of abortive transcription – important check-point in the regulation of gene expression

What are PROMTs?

o transcribed in reverse orientation to protein coding/sense sequence

o 0.5 – 1.5 kb long

o non-functional

o products of bidirectional promoters

 biproducts of transcription from protein coding genes

How does transcription termination regulate TSS-ncRNAs?

• Important for the regulation of promoter proximal pausing

• Important for inducible genes

• Every single polymerase starting from the promoer has the capacity to reach the end and produce mRNA

• If this is necessary, promoter-proximal pausing and abortive transcription will be overridden and polymerase will produce mRNA

• If it is not necessary, transcriptional termination will kick in to reduce transcription synthesis of full length mRNA

• Most polymerases will start and only produce short ncRNAs – very quickly terminated

What does transcription termination play a role in?

Gene activation and silencing

How does termination regulate PROMTs?

• Don’t have polyadenylation signals which trigger transcription termination of protein-coding genes in intergenic regions

• If ncRNAs are not terminated transcription may continue into regions located downstream

• In this situation, we have 2 polymerases transcribing towards each other (one for PROMTs, one for mRNA)

• Polymerase transcribing mRNA will be blocked by polymerase transcribing PROMTs

• Uncontrolled pervasive transcription results in transcription interference and is lethal

• Also creates a lot of tortion on DNA which may lead to DNA breaks and genomic instability

  

What are ncRNAs originating from nucleosome free regions (NFR)?

• Transcription from promoter of protein-coding gene results in mRNA and ncRNA

• Eukaryotic promoters are very often bidirectional and transcription is initiated in both directions (Sense or antisense direction)

Where can RNA Pol II initiate transcription?

at any NFR, not only from promoter

• Can happen at any place in introns or exons if the repositioning of nucleosomes is affected, eg in pathological conditions

• Observe transcription interference and deregulation of gene expression

How is transcription interference avoided?

• To avoid transcription interference and support proper transcription and expression of genes

• 2 complexes act on the promoters which are involved in transcription termination of ncRNA

• Integrator and restrictor (different from cleavage and polyadenylation complexes at 3’ end)

• paRNA – promoter-associated RNA

• TSS ncRNA – transcription start site ncRNA

• PROMPTs - promoter upstream transcripts

Take home message?

Non–productive transcriptional events at the protein-coding genes are immediately restricted to avoid transcriptional interference and deregulation of gene expression.

What is an example of ncRNAs processed from pre-mRNA intermediates?

snoRNAs

Where are human snoRNAs found?

Embedded into introns

What does synthesis of human snoRNAs depend on?

• Synthesis of human snoRNAs depends on expression of their host

• Very few human snoRNAs are independently expressed

• Many in the introns of protein-coding genes of long-coding RNAs

What does synthesis of snoRNAs in lower eukarotes depend on?

They are mainlu independent

How is snoRNA processed?

• When pre-mRNA is transcribed and spliced, if the intron contains snoRNA sequence, the sequence will be bound by specific snoRNA proteins

• When the intron is released and targeted for degradation, 5’-3’ and 3’-5’ exonucleases cant go through the structure because it is bound by snoRNA proteins

• It is therefore released to the nucleoblast

What are the 2 major classes of snoRNA?

boxC/D and boxH/ACA

What do boxC/D snoRNAs do?

RNA methylation

What do boxH/ACA snoRNAs do?

RNA pseudouridinylation

What is RNA methylation and pseudouridinylation required for?

required and essential for ribosome function and stability

What do snoRNAs recognise?

RNA targets via short complementary sequences

What are orphan snoRNAs?

snoRNAs with no known targets

How does the number of snoRNA units increase?

Increases with organism’s complexity

How many snoRNAs do we know in humans?

550 snoRNAs have been found

What is Prader-Willi syndrome?

• Neurodevelopmental disease

• autism-like syndrome

• the most common genetic disease causing morbid obesity

• fully manifests in early childhood

• no cure

• molecular processes leading to the syndromes are not known

What causes the PWS locus?

• Large deletion in chromosome 15

• Usually contains a lot of ncRNAs and protein coding genes

• Minimum deletion to cause PWS contains a cluster of orphan snoRNAs

• Some of these form human specific long ncRNAs called sno-lncRNAs

How do sno-lncRNAs form?

• 2 snoRNAs are in the same intron

• Exonucleases cannot go through snoRNA molecules

• End up with a long ncRNA with snoRNAs at either end instead of a cap and polyadenylate

• Makes it very stable

What do sno-lncRNAs do?

• sno-lncRNAs are one of the most abundant RNA species in human stem cells

• the intervening sequence may sequester splicing and transcription factors

• sno-lncRNAs regulate transcription of genes involved in neurodevelopment

• primate specific – not present in other species

Take home messages?

• snoRNAs functional and essential

• the balance between snoRNP assembly and degradation regulates their synthesis

• in higher organisms functions of many snoRNAs are still unknown

What are independently expressed ncRNAs?

• ncRNAs expressed independently of protein-coding genes

• transcribed from their own promoters

What are examples of Independently expressed ncRNAs?

o small nuclear RNAs (snRNAs)

o enhancer RNAs (eRNAs)

o long intergenic RNA (lincRNAs)

What are snRNAs?

• present in all eukaryotic organisms (U1, U2, U4, U5, U6)

• form riboprotein complexes

• required for splicing - essential

• short (~150 nt) and fold into “structural RNAs”

• non-polyadenylated, terminated by Integrator

• transcribed by RNA pol II apart from U6 which is transcribed by RNA pol III

What are enhancers?

• enhancers are cis-regulatory elements in the genome that cooperate with promoters to control target gene transcription

• active enhancers are highly transcribed

• are bound and mediated by mRNA specific transcription factors

What are enhancer RNAs terminated by?

terminated by either cleavage and polyadenylation complex or Restrictor or Integrator complexes

What are the 2 major types of eRNAs?

polyadenylated and non-polyadenylated

What are non-polyadenylated eRNAs?

bidirectionally transcribed eRNA of less than 2 kb (~350nt) terminated by Restrictor or Integrator complexes

What are polyadenylated eRNAs?

unidirectionally transcribed eRNA longer than 4 kb, transcribed from highly active enhancers, terminated by cleavage and polyadenylation, Restrictor or Integrator complexes

What are some suggested functions of eRNAs?

functional RNAs or by-products of transcription?

o Transcription keeps enhancers accessible for interaction with chromosomes

o OR

o eRNAs play roles in regulation of gene expression

What are the putative eRNA functions?

o Molecular glue keeping enhancer and promoter together

o Sequestering functions – bring transcription factors in proximity of promoters to enhance initiation processes

o Phase separated environment to facilitate gene expression

What diseases are eRNA mutations associated with?

• Cancers

  • eg breast cancer

  • Altered eRNAs transcribed from enhancers of genes like ESR1 (estrogen receptor 1) and MYC are linked to cancer progression and endocrine therapy resistance.

• Cardiovascular diseases

  • eg athersclerosis

  • eRNAs transcribed from enhancers of inflammatory genes (e.g., IL6, VCAM1) are implicated in vascular inflammation and plaque formation

• Neurodegenerative diseases

  • Alzheimer’s Disease

  • Dysregulated eRNAs involved in enhancer-promoter interactions of genes such as APP (amyloid precursor protein) may contribute to amyloid plaque formation.

• Metabolic diseases

  • eg Diabetes

    Dysregulated eRNAs associated with enhancers of genes like PPARG (key regulator of adipogenesis) and INS (insulin) may impair glucose homeostasis.

Take home messages?

• active enhancers are transcribed

• both transcription of the enhancer region and produced eRNA may be required for regulation of the target gene

What are long intergenic ncRNAs (lincRNAs)?

• Independent transcription units with defined start site, exons and introns, and termination site

• Longer than 200nt

• 13k transcripts arising from 9.5k gens

• Many functional and tissue-specific

• May evolve into functional mRNAs

lincRNAs vs mRNAs?

• synthesis employs the same processing machineries – capped, cotranscriptionally spliced and terminated in 3’ processes processed by cleavage and polyadenylation machinery

• processing of mRNA is much more efficient so is exported to the cytoplasm

• only some lincRNAs will be spliced and maturated so they are mainly in the nucleus

• many will be associated with the site where they are synthesised

What are lincRNAs functions?

lincRNAs functions are mostly associated with their high-order structure and/or ability to bind proteins (or DNA and RNA)

• Regulation of transcription

• Roles in nuclear organization by binding different parts of the genome

• Signalling

• Decoy/sequestering – making them accessible or inaccesiblle

• Guiding

• Scaffolding

What are examples of lincRNAs?

• MALAT1

  • regulates alternative splicing and modules gene expression

• Firre

  • interacts with chromatin and scaffolds epigenetic regulators

• HOTAIR

  • acts as a scaffold for histone modifying complexes

• As-Uchl1

  • regulates Uchl1 expression post transcriptionally and enhances its translation in response to stress

What do lincRNAs affect when removed from mice?

• Brain development

• Infertility

• Limb development

• Genome stability, ageing

• Immune system

• Viability

• Growth

Take home messages?

• lincRNAs are “non-coding copies” of mRNAs: are capped, spliced and polyadenylated

• lincRNAs are usually localized in the nucleus

• many lincRNAs are functional