RNA processing (13/14) Flashcards
What does the term RNA processing cover?
5’ end capping and 3’ end polyadenylation
Splicing
Editing
Surveillance (quality control)
Where does most of the processing happen?
Inside the nucleus where mRNA is present as pre-mRNA. This processing is specific to eukaryotes.
Heterogenous nuclear RNA (hnRNA)
pre-mRNA: RNA that compromises transcripts of nuclear genes made by RNA pol II. Only exists in eukaryotes.
Pre-mRNA processing (short summary)
The RNA is modified in the nucleus by additions to the 5’ and 3’ ends and by splicing to remove the introns. This is essential for the stability, export and translation of the mRNA strand.
5’ end capping (when it happens, simplified process, functions)
RNA capping generally occurs before further processing events. It happens as soon as the first 25-30 nucleotides are incorporated into the nascent transcript.
The process is catalyzed by various enzymes, such as RNA triphosphatase, gunylyltransferase and methyltransferase. Most mRNAs are modified by addition of 7-methylguanylate cap (m7G), which attaches to the phosphate bridge in an unusual 5’-5’ linkage involving 3 phosphates.
Functions:
- A capped mRNA is more resistant to 5’-3’- exonucleases, hence the de-capping stimulates mRNA degradation (decay).
- The cap is recognized by various protein factors (like the cap-binding complex CBC for export, or the translation initiation factor eIF4E) to facilitate: Splicing, export and translation (in the cytoplasm)
CBC
Cap-binding complex
3’ end polyadenylation (definition, where it happens, function, simplified process)
The addition of a poly-A tail (typically around 100-200 nucleotides) to a pre-mRNA. Happens both in the nucleus and in the cytoplasm.
It influences virtually all aspects of mRNA metabolism, conferring mRNA stability (protection from 3’-5’- exonucleases), promoting export and translation.
The process of polyadenylation is directed by a poly(A)-signal, on which the mRNA strand is cleaved. The cleavage is coupled to the addition of about 200 adenosines (As) to the 5’ cleavage product.
The polyA signal (in mammals)
Consists of the hexanucleotide AAUAAA and the G-U-rich elements. The cleavage occurs between these two elements.
Step 1 of 3’ end polyadenylation: RNA cleavage (proteins and auxilary cleavage factors involved)
The polyA signal functions as a landing platforms for different proteins.
- CPSF binds to AAUAAA
- CstF binds to the U-rich element
- CFI and CFII function as auxilary cleavage factors
CPSF
Cleavage and Polyadenylation Specificity Factor. Binds AAUAAA.
CstF
Cleavage Stimulation Factor. Binds the U-rich element.
CFI & CFII
Auxilary cleavage factors
Step 2 of 3’ end polyadenylation: Addition of the poly(A) tail
Once the cleaving occurs, the polyA tail is added to the mRNA. This addition is dependent on PAPs, which can be both canonical and non-canonical (in mitochondria).
As the poly-A tail grows, it associates with PABII for protection. Nuclear or cytoplasmic poly-A binding proteins depending on whether it is nuclear or cytoplasmic polyadenylation.
PAP
Template-independent poly(A) polymerase.
Canonical and non-canonical, e.g. in the mitochondria.
PABII
Nuclear poly(A) binding protein. Increases the PAP affinity to RNA, allowing efficient polyadenylation, and protects the RNA.
Alternative cleavage and polyadenylation (APA) (defintion, frequency)
APA occurs when a single gene has multiple polyA sites, and can result in multiple RNA transcripts and/or protein isoforms.
APA is widespread -> More than 70% of all human genes are subjected to APA.
Simple example of APA
There are 2 potential cleavage polyadenylation sites (2 possible cleavage events) in pre-mRNA. Which one is used depends on the associated proteins (like poly-A binding proteins) and additional proteins associated with the 3’ UTR.
Downstream is usually a strong poly-adenylation signal used to induce cleavage and addition of a polyA tail. This will result in a long 3’ UTR that has more potential for regulation.
Another upstream site will produce a very short mRNA when used. This will be too short to contain anything to regulate (no landing site). Various binding proteins (RBPs) or small RNAs like miRNAs will influence translation and stability of the mRNA (either repress or promote translation).
Various types of APA (general explanation, 3 scenarios, in-depth)
APA can produce a great variety of different mRNAs, but also at protein level depending on where the polyadenylation site is. If the alternative sites are in noncoding region, the protein doesn’t change. However, if they are within the coding region, then you also produce protein variants.
Contitutive polyadenylation: Single protein product. The 3’UTR will produce various mRNA variants depending on which of the cleavage sites the mRNA chooses. The protein, however, will not change.
UTR-APA: Single protein isoform, but protein output may vary between mRNA isoforms. Alternative cleaving and polyadenylation sites can interact with introns and exons. Introns can be maintained (instead of spliced) if there is a polyadenylation site within it. A poly-A tail added to this noncoding region makes it actually coding, which will produce a variant which is different in both the 3’UTR and also the protein sequence (with others than the usual exons).
CR-APA: Similar as above happens in alternative exons. Exons can be spliced in several different ways, which may produce a protein with extra bits and alternative 3’UTRs.
Pre-mRNA splicing (eukaryote/prokaryotes)
Excising introns and connecting the exons into a continous (pre)mRNA -> The protein-coding regions of pre-mRNA are joined together.
Mostly in eukaryotes, rare in prokaryotes. Here the genetic information is straight-forward and relates directly to the information translated. It becomes more complicated with splicing in for example mammals (gibberish has to be made into the actual code). Function of splicing is then to make sense of the code.
Relating number of introns with an organism’s complexity
There is a large variation within eukaryotes in number and distribution of introns. Complex organisms have intron-rich genes. Budding yeast has essentially no introns, whereas humans have 8 introns per gene.
Certain genes are very long but mostly consist of introns, e.g. the gene dystrophin with 98% introns.
Example of a spliceosomal RNA: U1 snRNA (Sm proteins)
All snRNPs, except U6, contain a conserved sequence that binds the Sm (Smith antigen) proteins.
Sm proteins are recognized by own antibodies (anti-SM) generated in an autoimmune disease (lupus erythematosus, SLE). These proteins are interacting with the Sm binding site in snRNAs and are involved in splicing.
RNA-RNA interactions that are key in splicing (+ what happens at the branch point?)
There are many interactions between the sequences in the splice sites that are mediated by the snRNPs.
snRNA engage in the interaction at the 5’ splice site by guiding the snRNPs’ interaction with the branch site. There are also some interactions between the snRNAs which are important for stability.
The branch site on the mRNA can be bound by either branchpoint binding proteins (BBPs) and later U2 snRNAs. This leads to the flipping out of the A which can engage in the nucleophilic attack.
The process of splicing (goal, sequence motifs, 2 transesterification steps)
Goal: To produce spliced mRNA which can be translated by discarding introns.
Splicing is mediated by special sequence motifs. An intron is framed in by a 5’ exon and a 3’ exon, with a 5’ splice site and 3’ splice site spanning the intron. Some of the residues in these motifs are conserved. Within the intron there is also a branch site A.
Splicing proceeds via 2 transesterification steps.
- Variant A in the splice site performs a nucleophilic attack on the phosphoryl group in the 5’ splice site. As a consequence, the intronic sequence is conformed into a conformation called a lariat (cowboy’s loop), which leaves a free 3’ hydroxyl at the end of the 5’ exon.
- The free 3’OH performs the second nucleophilic attack on the phosphate group in the 3’ splice site. The result is a lariat (spliced intron) and the joining of the two exons together. 2 phosphodiester bonds are broken, while 2 new are formed (doesn’t require energy).
Splicing is very energetically consuming, but it mostly comes from the spliceosome.
Spliceosome
The splicing workhorse. The spliceosome is a huge complex (ca. 12 MDa) containing many proteins and also some RNA: 5 x snRNAs (small nuclear). They also associate with particular proteins called snRNPs (“snurps”). In addition, there are some other core splicing factors and some auxilary proteins.
Most important are the snRNPs.
Biogenesis of snRNPs (snurps)
snRNAs are exported into the cytoplasm with the help of export factors. In the cytoplasm, they interact with various factors including Sm proteins which form (stabilizing) rings around the Sm sequence (Sm core assembly). With the help of other proteins, they go back into the nucleus and are processed in the cajal bodies, before they end up in the nuclear speckles.
Cajal bodies
Regions enriched in proteins and RNAs involved in mRNA processing. They are the main sites for the assembly of snRNPs.
True or false: Pre-mRNA processing occurs in the cytoplasm
False. Happens in the nucleus.
True or false: Splicing occurs mostly in eukaryotes
True. Very limited in prokaryotes
Senquence elements/motifs mediating splicing
5’ splice site, branch site, 3’ splice site
True or false: APA produces mRNA, but not protein, isoforms.
False. It can produce both.
True or false: Introns in different genes can vary in number and length.
True.
True or false: Like the ribosome, spliceosome consists of protein and RNA.
True. The most important proteins in the splicoesome are called snRNPs, and the RNA is called snRNAs.
Major vs. minor intron splicing (RNA elements/motifs)
The frequency of bases vary within the introns and splice sites in the RNA, e.g. in the branch site it is most likely that the sequence contains many cytosines. In the polypyrimidine track (only in major introns) there can either be a row of Cs or Ts.
There are some exceptions to this. There is a class of introns called minor-class introns, which are not so often found in mRNAs. They consist of slightly different motifs and don’t have a polypyrimidine track. Specific sequences are regulated with different factors.
Major vs. minor intron splicing (players)
The spliceosome is built up differently for major and minor introns, called type 2 and type 12 respectively. There are some differences in how these two classes of spliceosomes work.
The only common snRNP is the U5, while the others are different. The U11 and U12 is unique for minor and not found in major.
Major vs. minor intron splicing (mechanisms)
There is a big difference in the beginning of splicing.
In canonical splicing, there is one snRNP recognizing the 5’ splice site. Other factors recognize the branch site motif, which is later replaced by the U2 snRNP. There are also factors recognizing the polypyrimidine track, followed by a step-wise binding of U1 and U2.
Another key difference is that while USAF1 recognizes the branch motif in major introns, U11 and U12 can recognize the splice sites cooperatively, and function somewhat like a complex.
Types of introns and corresponding splicing mechanism (ribozymes)
tRNA and rRNAs = Enzymatic
Nuclear (pre-mRNA) = Spliceosomal
Group 1 = Self-splicing
Group II = Self-splicing
Key difference is that self-splicing doesn’t involve the spliceosome! Here, the introns function as ribozymes for self-splicing, which are RNAs displaying enzymatic activities.
Spliceosomal vs. self-splicing: group I and II introns
In the canonical pathway we find the familiar players (spliceosome, snRNPs), transesterification, flipping out A in the branch point sequence.
The group II self-splicing is a very similar pathway. Here there’s also adenosine with 2’ hydroxyl which performs a nucleophilic attack, followed by a second splicing event and the release of lariat, and finally the fusion of spliced exons. The key difference is that this reaction isn’t dependent on the spliceosome, but catalyzed by the intron itself.
The group I is quite distinct, as it doesn’t involve the spliceosome, but requires exogenous guanosine (exoG). This is coordinated by the intron, which is capable of catalyzing the first esterification reacion. The second big difference is that there is no lariat produced.
Alternative splicing (AS) (definition, common mechanisms)
Production of alternative mRNAs translated into various proteins. Increases the mRNA and protein diversity.
There are several common mechanisms of alternative splicing, including
- Alternative promoters: Differences in strength of the splice site. 2 different types of mRNA, 2 different proteins differing on the 5’ end N-terminus.
- Cassette exons: Exons which may or may not be incorporated into mRNA.
- Mutually exclusive exons
- Exon scrambling: Exons incorporated in different orders
- Alternative polyadenylation: 2 different proteins differing on the 3’ end C-terminus.
etc….
How can complex interaction influence splice-site selection? Enhancers/silencers
There can be alternative events depending on specific sequences and associated proteins: Exonic or intronic splicing enhancers (ESE/ISE) promoting splicing, or silencers (ESS/ISS).
One important family is the serine and arginine-rich SR proteins, which often function as splicing enhancers associating with the RNA enhancing sequences. Another family is heterogenous nuclear RNPs (hnRNPs), which often work as silencers/inhibitors of splicing.
The pattern and the ratio of these combinations of proteins vary from one cell to another and will determine the splicing outcomes. Depending on the availability of these factors, the patterns of splicing can be very different from one cell to another.
How is splicing co-transcriptional? (CTD, associated proteins during intiation/elongation/termination)
Splicing is co-transcriptional in the same way as capping and polyadenylation.
RNAPII has a long C-terminal domain (CTD) often phosphorylated at specific residues (serine 5 and serine 2). These tails are not only important for transcription, but also landing place for splicing.
During the initiation phase, CTD recruits capping enzymes, and capping happens very soon after the transcription starts. During elongation this is the place for the splicing factors. During termination phase, they are replaced by cleavage and polyadenylation factors.
Being co-transcriptional also means that splicing is affected by the chromatin.
Connections between chromatin and splicing (indirect/direct, active/passive, histone marks)
The DNA is never naked, always associated with proteins. DNA wrapped around nucleosomes can be modified, and the level of chromatin composition (closed/open conformation) may also affect splicing.
We can either have:
- Chromatin structure affecting splicing INDIRECTLY by altering the RNAP elongation rate. In open chromatin the pol II is transcribing fast, and might even favour exon skipping. Opposite in closed complex; pol II transcribes slowly, allowing more time for splicing factors to process alternative exons so that they might be incorporated. PASSIVE situation.
- DIRECT regulation by splicing factor recruitment to histone marks. A particular histone modification mark recruiting factors (like MRG15) associating with a certain splicing factor (PTB). These may process mRNA in a way with more exon skipping. In the absence of this histone mark, the exon isn’t skipped because MRG15 isn’t recruited. ACTIVE situation.
Not necessary to remember the factors!
Aberrant splicing and disease (RNA elements, 3 mutations/scenarios)
Aberrant splicing is often connected to lots of degenerate diseases in the brain, muscles etc.
3 types of mutations
- CIS-acting mutations, affecting a SINGLE gene.
- Trans-acting mutations, affecting MULTIPLE mRNAs.
- Nucleotide repeat expansions, affecting multiple mRNAs.
CIS-acting mutations: Aberrant splicing caused by mutations affecting a single gene (disease examples)
Mutation affecting the regulatory sequences for splicing (enhancers or silencers), leading to consequences in splice sites, branch points, or gain/loss of regulatory sequences. Depending on the mutation type, there are various types of associated diseases. In mRNA subjected to splicing, there is only one gene affected.
Diseases: Spinal muscular atrophy, CF, Duchenne muscular dystrophy, cancer
Trans-acting mutations: Aberrant splicing caused by mutations affecting multiple mRNAs (disease examples)
More complicated when a mutation affects the facultative or consequtive factors affecting splicing (snRNPs, enhancers/silencers). These mutations have an effect of factors splicing many different mRNAs, making them difficult to treat because of their many targets.
Diseases: Spinal muscular atrophy, autism disorder, Alzheimer’s disease, Huntington’s disease
Nucleotide repeat expansions: Aberrant splicing caused by mutations affecting multiple mRNAs (disease examples)
Nucleotide sequences that can enhance can often lead to production of a structure with the potential to recruit various DNA binding proteins like splicing regulators (SF). This might result in titrating splicing factors away from the site where they should be working (sinks for the splicing factors), resulting in loss of splicing function. Will recruit or repel splicing factors functioning on multiple mRNA targets
Diseases: Myotonic dystrophies 1 and 2, Huntington’s disease
Aberrant splicing and disease: Therapies (examples)
Some of the diseases caused by aberrant splicing can be treated with synthetic therapeutic nucleotides pairing with specific elements.
Therapeutic goals: Exon retention
Spinal muscular atrophy (single gene): The gene gets mutated to contain an element resulting in exon skipping, producing non-functional protein resulting in disease. If you target a specific silencer by a nucleotide you can avoid the silencing and restore the protein function.
Duchenne muscular atrophy (single gene): Part of sequence targeted is a mutated exon. If you target the splicing of this exon in a way where it isn’t retain in the mRNA, you can cure the patient.
Myotonic dystrophies 1 and 2 (multiple genes): Disease comes from the titration of the MBLN protein involved in splicing. You can prevent the titration with synthetic CUG repeats to release these splicing factors so that they’re able to perform their jobs.
mRNA editing (definition + 2 types)
Further modification of pre-mRNAs prior to nuclear export.
Deamination of bases leads to codon changes, and two different processes are C -> U editing and A -> I editing.
Example of C -> U editing
If you change the C to an U in the coding mRNA, you can change codons depending on where this editing occurs. This kind of editing happens in mRNA encoding apolipoprotein B (important for lipid metabolism). You have a long and short version of the protein. The short version is faster, whereas the long version will result in buildup of lipids in the bloodstream. Important to have regulation of ratio between long and short version by editing into the short version by converting CAA into UAA (stop codon).
Example of A -> I editing
Editing regulates the activity of glutamate receptors (whether the proteins are inactive or active). The glutamate receptor is essential for communication in the nervous system.
The encoding gene produces pre-mRNA with strucutre elements recruiting ADAR2 enzymes, which changes A to I to effectively change the codon from CAG to CGG (because I pairs with C in tRNA). Instead of encoding glutamine, it now encodes arginine.
Only the edited form of the protein is functional. This is therefore an example of how editing is essential, since the protein encoded by the genome is useless and can lead to death. Why wouldn’t you then just encode the functional protein?
Pre-mRNA surveillance: 5’ capping and transcription elongation
Unprocessed mRNA are unprotected and subjected to degradation by exonucleases. This can happen during the 5’ end formation. Thus, capping is essential, with certain proteins with CBC associating and protecting.
If there is delayed or aberrant capping, mRNA will be degraded by 5’-3’- exonucleases with catalytic site Xrn-2 (like pacman) -> It first removes phosphates at the end, following by degradation nascent RNA.
Following cleavage you will have unprocessed mRNA which can cause problems during elongation if it is premature. RNAPII will stall and backtrack, letting the 3’ end of mRNA be expose, which will subject it to degradation by 3’-5’ exonucleases.
Pre-mRNA surveillance: Polyadenylation & splicing
Possible issues may arise during splicing. The spliced out lariat following dissociation of the spliceosome will be subjected to degradation, which will produce a delayed or aberrant splicing with an unprotected 3’ end subjected to 3’-5’ exonucleases. The mRNA therefore has to be immediately associated with polyadenylation to avoid this.
Generally, the spliceosome splices out …
Introns of pre-mRNAs
In contrast to the major splicoesome, the minor spliceosome …
Contains the U12 snRNP (sometimes called the 12-path because of this unique snRNP)
Group I introns are spliced …
Using an exogenous G (no lariat)
Alternative splicing …
Greatly increases proteome complexity
Like the pre-mRNA end processing, splicing is …
Co-transcriptional
Trans-acting mutations affecting splicing impact …
Multiple mRNAs (not easy to fix, but possible)
As the consequence of mRNA editing …
Certain codons become re-coded
Aberrant 5’ end capping results in …
mRNA decay, mediated by 5’-3’ exonucleases
Correctly processed mRNA is protected from the 3’-5’- nucleases by …
The polyA tail and associated PABs (poly-A binding proteins)
