IT5: RNA processing Flashcards
How are mRNA transcripts modified in eukaryotes?
- 5’ Capping
- Splicing
- Polyadenylation
- 3’ Cleavage
Describe the following RNA types:
- eRNA
- miRNA
- siRNA
- snoRNA
- snRNA
- piRNA
- lncRNA
eRNA: transcribed from enhancer DNA, it’s thought to regulate transcription in cis and trans.
miRNA: RNAi (endogenous)
siRNA: RNAi (exogenous)
snoRNA: guide RNA modifications
snRNA: form snRNPs to process pre-mRNA (e.g., spliceosome)
piRNA: TE suppressors
lncRNA: >200nt with various roles e.g., Xist
Which type of RNA is most prominent in cells by mass, and by number of molecules?
Mass = rRNA
Number = tRNA
Do prokaryotes process their RNA? If yes, how?
Bacterial cells do process, but not extensively as transcription and translation are coupled and so there’s less of a necessity to do this.
As tRNA and rRNA are the main stable RNAs found in prokaryotes, these are usually processed.
How is rRNA processed in eukaryotes?
rRNA is processed in the nucleolus by RNAPI. pre-rRNA is a large molecule that must be cleaved and trimmed at introns via snoRNA-mediated modifications to create the individual ribosome subunits.
How is tRNA processed in eukaryotes?
- Trimming by exonucleases
- Splicing by endonucleases to remove the sequence occluding the anticodon
- CCA synthesis at the 3’ end
- 3’OH and 5’P synthesis and ligation
- Modifications
What’s the difference between endo- and exonucleases?
Both catalyze the hydrolysis of phosphodiester bonds in DNA and RNA.
Endo: cleaves within the DNA or RNA molecule
Exo: cleave the last nucleotides of DNA or RNA
How do we know that transcription and processing are tightly linked in eukaryotes?
Some components of the processing machineries can interact with general transcription factors and thus are recruited to the PIC.
e.g., TFIIH interacts with the 5’ capping machinery.
What is the importance of Rpb1, the largest RNAPII subunit, in pre-mRNA processing?
What is the evidence for this?
Experiments showed that deletion or shortening of the Rpb1 CTD has dramatic impact on pre-mRNA processing efficiencies, both in vivo and in vitro.
The CTD contains a heptapeptide sequence, YSPTSPS, where Ser2 and Ser5 are phosphorylated by Cdks for RNAPII function.
What is the significance of phosphorylating Ser5 and Ser2 of the Rpb1 CTD?
Ser5: allows elongation to start. The CDK responsible for this is part of the TFIIH machinery, so phosphorylation shows that TFIIH is ready.
Ser2: recruits splicing machinery and releases RNAPII for elongation after capping has occurred.
When and how does 5’ capping occur? How does this trigger elongation?
(Start from Ser5 phosphorylation)
Ser5 of the Rbp1 CTD is phosphorylated by TFIIH, remodeling the complex for elongation.
Elongation factors are recruited to the CTD to arrest RNAPII for capping to occur.
Capping machinery is recruited. Triphosphatase enzyme removes the terminal phosphate, guanylyltransferase adds a GMP (forming GpppN) and methyltransferase forms the final m7G cap structure.
Once capping is complete, elongation factor pTEFb phosphorylates Ser2 which allows elongation to commence.
What is the cap binding complex?
Heterodimeric protein that binds the m7G cap structure.
CBC20 binds Ser5P
CBC80 binds Ser2P
It helps protect the mRNA from degradation and promotes efficient splicing.
What is pTEFb and how is it activated?
pTEFb is a heterodimeric protein complex composed of a cyclin-dependent kinase (CDK9) and a regulatory cyclin subunit. pTEFb is a critical regulator of RNAPII transcriptional elongation, as it phosphorylates the CTD of RNAPII to promote productive elongation of the RNA transcript.
Its activity is tightly controlled by stress response proteins and proinflammatory signals.
Describe the spliceosome. How does it interact with pre-mRNA?
A large complex made up of snRNAs - U1/2/4/5/6snRNA - and many other proteins to form snRNPs.
It binds the Ser5P.
Describe the mechanism of pre-mRNA splicing.
- U1snRNA identifies the 5’ splice site
- U2snRNA binds proximal intronic branch point (next to 3’ splice site)
- Other snRNPs bind to make the intron loop and bend.
- Branch point nucleotide carries out nucleophilic attack on 5’ nucleotide.
- Intron is cleaved from upstream exon
- 3’OH on upstream exon undergoes nucleophilic attack on the 3’ splice site and fuses the two exons together.
What is meant by ‘proximal intronic branch point’? Why is it important?
A sequence element located 18-40 nucleotides upstream of the 3’ splice site in the intron. This is recognized by U2snRNP to aid in intron removal.
This is an important element because variations in its sequence or distance from the 3’ splice site can influence efficiency and accuracy of splicing.
[Usually adenine residues]
How is transcription termination linked to mRNA cleavage and polyadenylation?
Describe this process.
When RNAPII synthesizes past a poly(A) site, AAUAAA, this signals for cleavage ~30bp further along. The AAUAAA cleavage signal is bound by CPSF (cleavage and polyadenylation specificity factor) which has endonuclease activity to cleave the pre-mRNA.
Poly(A) polymerase (PAP) adds the poly(A) tail to the 5’ end, which is subsequently covered by poly(A) binding proteins.
The 3’ cleaved end of the pre-mRNA remains attached to RNAPII for exoribonuclease degradation. This forces mammalian RNAPII to terminate, known as the torpedo model. This is followed by the phosphorylation of CTD to recycle it for another round of transcription.
What is the torpedo model of RNAPII termination?
Transcription termination occurs through the action of an exonuclease, which degrades the RNA transcript in a 5’ to 3’ direction after CPSF-mediated cleavage.
The exonuclease is thought to act as a “torpedo” that chases after the RNAPII complex that is still bound to the RNA transcript. As the exosome degrades the RNA molecule in a 5’ to 3’ direction, it catches up to the RNAPII complex and causes it to dissociate from the DNA template.
How is capping linked to promoter proximal pausing, premature transcription termination, and transcriptional pause release?
PPP: Capping is thought to be necessary for RNAPII to pause and accumulate near the promoter region of the gene. This pausing provides a mechanism for regulating gene expression, as it allows the cell to quickly activate or repress transcription in response to signals or stimuli.
PTT: Premature termination is thought to be linked to capping because the cap structure is necessary to protect the 5’ end of the RNA from exonucleases that could degrade the mRNA before it is fully transcribed. Without capping, the mRNA is more vulnerable to degradation, making premature termination more likely.
TPR: Capping recruits elongation factor pTEFb which uses its CDK activity to phosphorylate Ser2 and allow elongation to occur.
What is the downstream sequence element, involved in pre-mRNA cleavage?
The downstream sequence element (DSE) is a cis-acting element in pre-mRNA that is recognized by the CPSF.
Once CPSF binds to the DSE, it recruits other factors involved in the processing of the 3’ end of the mRNA, including the cleavage stimulation factor (CstF) and the poly(A) polymerase (PAP).
What is the importance of alternative splicing? Give an example of a highly alternatively spliced gene.
It increases protein diversity without increasing DNA content and keeps the ORF intact. e.g., Dscam in Drosophila can generate over 38000 isoforms of the gene.
What are SR proteins and how are they involved in pre-mRNA splicing?
SR proteins (serine/arginine-rich proteins) are a family of RNA-binding proteins that play a key role in regulating pre-mRNA splicing.
SR proteins bind to specific sequences on pre-mRNA transcripts called exonic splicing enhancers (ESEs) and promote the recruitment of the spliceosome machinery to these sites.
What is the exon definition model?
According to the exon definition model, the recognition of exons is based on the interaction of splicing factors with specific cis-acting elements located within the exons themselves. These elements include exonic splicing enhancers (ESEs) and exonic splicing silencers (ESSs), which recruit either splicing activators or repressors, respectively, to the exon.
Hence, splicing is dependent on the context of the surrounding exons to the splice sites, rather than the introns. This makes sense, seeing as even small introns can be spliced out with efficiency, despite being theoretically hard to recognize.
What are exonic splicing enhancers and silencers, and what binds to each of these?
ESE: recruits SR proteins to increase splicing
ESS: recruits hnRNPs to block splicing
What is the importance of splicing in Drosophila sex determination?
Sxl is encoded on the Drosophila X chromosome, so only females (XX) have high enough doses of it.
Sxl is an alternative splicing inhibitor, meaning that it blocks one splice site from being recognized and so splicing occurs further downstream. This produces functional proteins for female sex differentiation.
Without Sxl blocking the site, a different splicing pattern will occur that produces non-functional proteins.
How is alternative splicing regulated?
- DNA methylation
CTCF-bound DNA acts as a block, slowing RNAPII so weaker splice sites can be recognized. When the CTCF binding sites are methylated, it prevents CTCF from binding so RNAPII doesn’t slow. - Histone modifications
Different histone modifications can recruit splicing factors, such as the polypyrimidine tract binding protein (PTB).
What is alternative polyadenylation and why is it important?
It involves the selection of different poly(A) sites during mRNA processing, resulting in the production of mRNA transcripts with different 3’ untranslated regions (UTRs) and, potentially, different protein-coding regions.
It can contribute to the expansion of the proteome.
How is alternative polyadenylation used in antibody class switching?
B cells can switch between different classes of antibodies (e.g., IgM, IgG, IgA) through a process known as class switching. During class switching, the constant (C) region of the antibody heavy chain is recombined with a new downstream C-region gene segment, resulting in a new antibody class.
In B cells, alternative polyadenylation occurs at a gene called Cα, which encodes the constant region of the IgA antibody. The Cα gene contains multiple poly(A) sites, which allow for the production of mRNA isoforms with different 3’ UTRs. 3’ UTR length is responsible for some class switches.
How is alternative polyadenylation used in cancer cells?
By shortening their 3’UTRs, cancer cells stand a greater chance at not having mRNA degraded by miRNAs.
How can alternative polyadenylation impact mRNA localization?
The 3’ UTR contains binding sites for regulatory factors that can affect the stability, translation, and localization of the mRNA. Therefore, changes in the 3’ UTR length resulting from APA can alter the binding sites for these regulatory factors and thereby affect the fate of the mRNA.
For example, some studies have shown that shortening of the 3’ UTR through APA can increase the stability of the mRNA and enhance its translation. This may occur because shorter 3’ UTRs have fewer binding sites for miRNAs, which are known to destabilize and repress translation of target mRNAs. Alternatively, longer 3’ UTRs resulting from APA may contain additional binding sites for RNA-binding proteins that can influence the localization of the mRNA.
How is alternative polyadenylation regulated? Give an example.
Differential expression of core poly(A) factors and other RNA binding proteins that regulate poly(A) site recognition.
E.g., by blocking the AAUAAA recognition site, or making it less like the consensus, you can block alternative polyadenylation.
What diseases are associated with pre-mRNA capping?
- Influenza - ‘cap snatching’ from host mRNA is used to provide viral polymerase with an RNA primer.
- Herpes simplex virus - inhibits CDK9 phosphorylation of Ser2, stalling polymerases.
What diseases are associated with pre-mRNA splicing mutations in cis-elements?
- Alzheimer’s - alternative splicing is important in balancing ratios of tau proteins, so mutations in this can cause aggregation.
- DMD - known mutation arises in ESS formation that causes exon skipping.
- Cancer - KIT tyrosine kinase has a cancer mutation that can cause splicing out of the control region, making it active at all times.
How can mutations arise in the different phenotypes observed in Duchenne muscular dystrophy caused by either T to A substitutions in exon 31 vs. abolishment of Tra2B binding?
T to A substitution: there is interference with splicing, but Tra2B can still bind to ‘boost’ splicing occurrence.
Abolishment: Tra2B can’t aid in splicing at all, so phenotype is much worse.
What diseases are associated with pre-mRNA splicing mutations in trans-elements?
- Prader-Willi syndrome - deletion of a gene that encodes snoRNAs. Normally these snoRNAs contributes to serotonin receptor gene expression, but a lack of these causes skipping of exons in these genes and thus truncated receptors that cannot be activated.
What diseases are associated with poly(A) signal mutations?
Ocular muscle dystrophy:
Mutations cause extension of the poly(A) binding protein ORF that results in expansion of the poly(A) stretch. This causes aggregation of PABs in the nucleus.
Cancer:
Cancer cells use shorter 3’UTRs to evade miRNA targeting for degradation.
What are RNA therapeutics? Give examples.
RNA therapeutics are a class of drugs that target RNA molecules for therapeutic purposes. They are designed to modify the expression of genes by targeting specific RNA molecules involved in disease processes.
e.g., mRNA vaccines to trigger immune responses (they require modifications to reduce immunogenicity).
RNAi drugs can target specific disease-causing genes and inhibit their expression. e.g., Patisiran which targets the TTR gene, causing a rare genetic disorder due to accumulation of proteins in tissues and organs.
What are the advantages and disadvantages of RNA therapeutics?
+ Fast acting
+ Well-tolerated
+ High specificity
+ Can target previously ‘undruggable’ targets
- Short-lived expression
- Typically requires IV administration
What are the ribosomal subunits/sites that bind the:
- Shine Dalgarno sequence
- AUG start codon
SD: 30S
AUG: P-site
How can the m7G cap be further modified?
Cap methylation, which involves the addition of one or more methyl groups. These modifications can affect mRNA stability, translation efficiency, and interactions with other cellular proteins.
What translation initiation factors associate with the CAP binding complex?
eIF4A/E/G that form the eIF4 complex (eukaryotic initiation factor).
What is the function of each of the subunits in the eIF4 complex?
eIF4A: ATP-dependent unwinding of the 5’ end of mRNA
eIF4E: actual cap binding protein
eIF4G: scaffolding protein that binds the other two subunits
Which complex of proteins does the 43S ribosomal subunit recognize to find AUG?
By binding eIF4 complex that’s attached to the cap. 43S then scans the 5’UTR to locate an AUG start codon.
Why is correct scanning to find AUG in eukaryotic mRNA so important?
Eukaryotic ribosomes associate with the CAP, not the starting codon, and so it has to cross the 5’ UTR sequence before reaching the AUG. Thus, the P site isn’t directly occupying the AUG. This means that the ribosome must ‘scan’ the mRNA until it finds the starting codon before it recruits the 60S subunit.
This is critical because only the correct AUG will arise in the correct ORF and thus, a functional protein.
Which two events undergo phosphorylation-dependent regulation to control protein synthesis?
- Formation of the ternary complex
- Loading of ribosomes onto mRNA
Describe how the formation of the ternary complex is regulated by phosphorylation.
The ternary complex involved in translation initiation consists of eukaryotic initiation factor 2 (eIF2), GTP, and methionyl-tRNA (Met-tRNAi).
In its unphosphorylated state, eIF2 binds GTP and delivers Met-tRNAi to the ribosome to initiate translation.
Phosphorylation of eIF2α at Serine 51 results in a conformational change that prevents eIF2 from binding GTP and blocks the delivery of Met-tRNAi to the ribosome, thereby inhibiting translation initiation. This process is a critical part of the cellular stress response, as it helps to conserve energy and resources in response to various stressors.
How is eIF2 related to diabetes?
In diabetes, high levels of glucose and insulin signaling can increase eIF2 phosphorylation, preventing ternary complex formation and translation from occurring.
This reduces protein synthesis and can impair insulin signaling.
How is loading of the ribosome on to mRNA regulated by phosphorylation?
One of the eIF4 subunits (eIF4e) can be phosphorylated in response to growth promoting signals. This promotes eIF4 complex formation on the 5’ caps of mRNA, although it’s not clear how this impacts phosphorylation.
This can be blocked by 4E binding proteins.
How does mRNA function as a ribonucleoprotein?
mRNA can contain sequences that can interact with proteins to form ribonucleoprotein complexes. e.g., eIF4 complex binding to the 5’ cap.
What causes mRNA to become circular? Why is this important?
Interactions between poly(A) binding proteins and the cap binding complex.
Both the 5’ and 3’ UTR are involved in translation regulation at the initiation stage, so bringing these sequences together is important for getting this right.
What are TOP mRNAs? Why are they tightly regulated?
Group of mRNAs that contain a characteristic 5’-terminal oligopyrimidine (5’TOP) sequence of 4-15 pyrimidines (uracil and cytosine) followed by a purine (adenine or guanine) and then an AUG translation initiation codon.
These mRNAs encode ribosomal proteins and translation factors, which are required for protein synthesis. This motif allows for accelerated ribosomal loading.
The regulation of TOP mRNA translation is important for the control of protein synthesis and cell growth, and dysregulation of TOP mRNA translation has been implicated in various diseases, including cancer and metabolic disorders.
How are intracellular iron levels regulated? Why is this important?
Intracellular iron levels are regulated by iron-responsive elements (IREs) in mRNA. IREs are stem-loop structures found in the untranslated regions (UTRs) of certain mRNAs that encode proteins involved in iron metabolism.
These are used to regulate iron levels in 2 ways:
1. High iron causes the blocking of iron responsive proteins from binding IREs on iron storage proteins, allowing these proteins to be expressed.
2. High iron also causes 3’UTR recognition of genes encoding transferrin receptors to prevent more of these iron transporters from being made.
How do features within mRNA aid in dosage compensation triggering in Drosophila?
Dosage compensation is only needed in male Drosophila due to their lack of an extra X chromosome.
To prevent this pathway from occurring in females, the XX-dependent protein SXL binds both the 5’ and 3’UTR of the MLS2 gene to prevent translation.
MLS2 is involved in triggering the dosage compensation pathway.
How can maternal mRNAs be regulated using CPEB?
CPEB (Cytoplasmic polyadenylation element-binding protein) is a key regulator of maternal mRNA translation in oocytes and early embryos.
Oocyte maturation: CPEB binds 3’UTR and triggers lengthening of the poly(A) tail to act as a binding site for translation initiation factors.
After fertilization, CPEB is degraded which shortens the poly(A) tail and inhibits translation. This ensures that only maternal mRNAs required for early development are translated.
How can viruses use IRES motifs for cap-independent translation initiation?
Viruses can use internal ribosome entry site (IRES) motifs to promote cap-independent translation initiation of their mRNA.
IRES motifs are RNA structures found in the 5’ UTR that allow ribosomes to bypass the need for a 5’ cap structure and bind directly to the mRNA.
Why is seleno-cysteine incorporation at some stop codons important?
Selenocysteine (Sec) is a rare amino acid that is co-translationally incorporated into proteins in response to a UGA codon.
Proteins that contain Sec are called selenoproteins and play important roles in various biological processes, including redox regulation, thyroid hormone metabolism, and immune function.
What is deadenylation-dependent and -independent degradation?
Dependent: cap and poly(A) tail are removed by specific enzymes for mRNA degraded by exonucleases.
Independent: mRNA is cleaved in the center so cap and tail removal isn’t needed for degradation.
How are AREs related to mRNA half-life?
AU-rich elements are destabilizing sequences found in the 3’UTRs to recruit decay machinery.
What is the exon junction complex? What is its function?
When splicing occurs, exons are fused together arising in an exon junction. During splicing, there’s a number of proteins deposited at the exon junctions, known as the exon junction complex.
This is important because it shows the ribosome that exon ligation has occurred properly after splicing. If an EJC doesn’t form at an exon junction, it signals that something has gone wrong.
It’s thought that the ribosome synthesizes a sort of ‘trial run’ mRNA which allows it to recognize the lack of EJCs, etc. If it does find an EJC missing, it causes decay of the mRNA.
What is the epitranscriptome? What is the most common modification?
The epitranscriptome refers to the collection of various chemical modifications that occur on RNA molecules, particularly messenger RNA (mRNA), after they have been transcribed from DNA.
- m6A which influences alternative splicing and mRNA decay
What are karyopherin proteins?
Importins and exportin proteins, involved in the Ran-importin pathway.
How is mRNA exported from the nucleus?
After processing, the mRNA is bound by a series of proteins including the cap-binding complex and exon junction complex, that facilitates the assembly of the mRNA export complex: TREX.
TREX interacts with the nuclear pore complex for transport, and disassembles in the cytoplasm.
What is so special about yeast and their mechanisms for mRNA export?
Yeast contain proteins such as SAGA that react to a gene being turned on, and move it from the center of the nucleus to the periphery so that mRNA movement into the cytoplasm is much more efficient.
Give examples where mRNA localization is important.
- oskar mRNA localization determines proper abdominal formation in Drosophila
- Neurons - these have longer 3’ UTRs that undergo AP to achieve polarity
- Migrating fibroblasts - mRNA is localized to focal adhesions to regulate adhesion
What are paraspeckles?
Paraspeckles are dynamic nuclear bodies found in mammalian cells that are formed through the assembly of long non-coding RNAs (lncRNAs) and RNA-binding proteins.
They form in response to stress and help to sequester proteins and RNA.
What are P-bodies and stress granules?
P-bodies: degrade mRNAs and miRNAs, as well as being involved in mRNA storage and translation repression.
Stress granules: formed in response to stress to regulate mRNA translation by sequestering mRNAs and translation factors.
What are germ granules?
Membrane-less organelle that harbors RNA and proteins critical for germ cell development.
Describe the experiments that discovered miRNAs using C. elegans genetic experiments.
Let-7 was shown to be a lethal mutant for C. elegans and encoded a long RNA molecule.
This mutation could be suppressed by another mutation in lin-41.
We now know that let-7 encodes miRNA that targets lin-41 for degradation. When lin-41 was knocked out with let-7, less lin-41 was being produced anyway so the mutant wasn’t lethal.
Describe the process of miRNA biogenesis.
- Transcription, resulting in a transcript that contains a hairpin structure
- Drosha cleaves the pri-miRNA at the base of the hairpin, forming pre-mRNA
- pre-mRNA is exported to the cytoplasm and cleaves to form RNA duplexes by Dicer
- The duplex is loaded onto a RISC which contains an Ago protein
What gives Ago proteins their slicer activity?
Their PIWI domains
What is the SEED sequence?
A short sequence in miRNAs that binds to the 3’UTR of target mRNAs for translational repression. Whilst the rest of the sequence doesn’t need to be complementary, the SEED sequence must.
Why do most miRNA deletion mutants not show significant phenotypic changes?
- Redundancy between miRNAs
- miRNA acts as a dampener of target mRNA fluctuations, rather than to completely degrade it
What are piRNAs?
PIWI-interacting RNAs, are a type of small non-coding RNA that play a role in regulating gene expression and maintaining genome integrity in the germ cells of animals.
They are typically 24-32 nucleotides in length and are associated with a specific class of proteins called PIWI proteins, which belong to the Argonaute protein family.
Where are piRNA genes found? How are they transcribed and exported?
Heterochromatin, marked by H3K9me3. This repression is mediated by Rhino, a HP1 homologue.
TRF2 is part of a chromatin remodeling complex that can interact with Rhino to promote the recruitment of RNAPII and facilitate piRNA transcription.
Exportation requires a specific pathway because piRNAs aren’t spliced.
What are primary and secondary piRNAs?
Primary piRNAs are generated from long single-stranded precursors transcribed from piRNA clusters.
Secondary piRNAs, on the other hand, are produced from cleavage of target transcripts by the RNA-induced silencing complex (RISC) containing primary piRNAs. This process is known as the ping-pong cycle, where the cleaved target RNA serves as a substrate for RISC to generate new piRNAs.
What are siRNAs and how were they discovered?
siRNA stands for small interfering RNA, which is a type of RNA molecule that plays a role in gene regulation via RNAi.
Discovered first in plants when it was noticed that plants are able to prevent the spread of viruses. Later, this same pathway was found in C. elegans were it was noticed that injecting dsRNA into the nematodes causes highly effective silencing.
