Lecture 21 (RR9): RNA Processing II Flashcards
rRNA processing
rRNAs also undergo processing:
* A lot of class II genes are spliced. Other classes of RNA are also processed but not spliced in the same way (do not require a spliceosome).
* rRNAs arise from arrays of ribosomal DNA.
* Transcription always occurs in the same sequence: you will generate the same rRNAs from that transcriptional process: 18S, then 5.8S, then 28S. All processed from a large rRNA molecule with limited conservation. It is the sequence that seems to be very important (conserved from yeast to humans).
* Process requires snow RNAs.
* The introduction of an rDNA into a cell is sufficient to set up a nucleolus (membraneless organelle that exist in the nucleus) → Nucleoli are liquid liquid condensates within the nucleus formed by the transcription of the rDNA copies.
What are the tRNAs modifications?
Pre-tRNAs are transcribed (as precursors) by RNA Pol III and processed to yield mature tRNA
1) the 5’ end sequence is removed (in green)
2) a short segment is removed (in blue) to give rise to cloverleaf structure
3) CCA is added on to the 3’ end (critical for quality control - assure tRNA folds up properly and for charging of tRNA with its proper amino acids
4) extensive modification of internal bases
Only class II RNAs that are generated by RNA polymerase II get spliced using the spliceosome as described.
Self-splicing group II Introns
Self-splicing group II introns form structures resembling the spliceosome
* Group II introns are now only present in mitochondria and chloroplast genes, but they may be the evolutionary predecessors of other introns
* Self-splicing introns were the first evidence that RNAs can carry out catalysis on their own
* Carry out trans-esterification reactions independently from protein
* Proteins may have been a later addition to RNAs in evolutionary time
RNA binding domains
RNA-binding proteins that share the same motif (RRM domain):
* Many of the components that are required for correct splicing don’t necessarily have a small nuclear RNA component.
* A number of key proteins that are required for efficient and reproducible splicing are mediated by RNA binding domains.
* One of the best RNA binding domains is the RNA recognition motif (also called the RRM domain) → has 2 highly conserved regions (RNP1 and RNP2).
- Proteins will interact in a specific and non-specific manner to RNAs
a) RNA recognition and Sex-lethal: interacts with RNA through beta sheets that are positively-charged – positive regions interact with negative regions of the RNA
b) PTB: Positive regions interact with nucleic acids - Other RNA binding domains: KH domains, RGG repeats
- Confer some ability to bind to RNA
Exons
- Exons are generally quite small relative to introns in higher eukaryotes (150bp vs 3500bp)
- Some introns can be over 500kb
- Exonic splicing enhancers are sequences within the exon that promote exon joining during splicing → decorate exons
- At the 5’ end of the intron to be excised, the U2 snRNP recognizes where to sit with the help of U2AF
SR Proteins
- Rich in serine and arginine, they have disordered domains (important for forming condensates).
- RNA binding proteins with RRM domains and protein:protein interaction domains
- Bind exonic splicing enhancer sequences in exons
- Facilitate binding of the U1snRNP to the true 5’ splice site and the U2snRNP to the branch point
- SR Protein:protein/snRNA interactions for the cross-exon recognition complex
- Whenever you hear about intrinsically disordered domains, think about: SR proteins interact with sequences within exons → referred to as exonic splicing enhancers, they decorate the exons.
U2AF
U2AF – a protein component that helps with splicing efficiency → works with SR protein to define where the intron exon boundary is.
* Small subunit binds to AG dinucleotide at the 3’ end of the intron to be excised – it helps to define that boundary (it recognizes the SR proteins).
* U2AF will recognize AG dinucleotide because a number of SR proteins decorates the exon by interacting with specific sequences in the RNA that defines the exon called exonic splicing enhancers
* Larger subunit interacts with polypyrimidine track in the intron – it helps the U2 snRNP recognize the branch point and sit down on the branch point correctly.
What happens if these proteins are not regulated (SR, U2AF)…
- All these proteins (SR proteins, U2AF..) are critical for ensuring that each time an intron is removed, you remove the correct sequence and all of that sequence
- If you leave bits of introns, you get an inframe stop (frame shift in the protein). Cause havoc or is lethal.
- These proteins have to be regulated, this regulation is at the heart of alternative splicing: how you can splice RNAs in different manners depending on temporal information or tissue specific.
- Through evolution, we discovered that one single transcription unit can give rise to multiple different gene products. You can make lots of different proteins out of a single RNA that is spliced up differently with different exons.
Common example of alternative splicing
Fibronectin
* Fibroblast (make up skin) use fibronectin to adhere the cells to specific substrates, it is sticky due to the two green exons (EIIIA and EIIIB).
* Hepatocyte (liver cells) secretes into the bloodstream and you don’t want it to be sticky in this case (blood clots) so it does not have the two green exons.
Alternative splicing and drosophilia
- Alternative splicing controls sex determination in Drosophila
- Mixing and matching of exons
- Clear differences between male and female flies
- There is clear sexual dimorphism which is a reflection of the chromosome content = Dosage compensation
- Males have smaller abdomen
Control of Sex-Lethal Expression
- Gene that is critical for compensating for either the double dose of the X chromosome or the lack of an X chromosome → dosage compensation
- Downstream of a chromosome counting mechanism very early on
- It will bring about changes that will be reflected in the sexual dimorphic outcomes of the developing organism
- Sxl is under transcriptional control; it is expressed only in females in early embryogenesis → from a promoter that is only active in females (only females make sex lethal protein).
- It makes changes by interacting with specific regions of pre-mRNAs
- Later in development, the female-specific promoter is repressed and a different Sxl promoter is activated that is on in both sexes
- However, Sxl pre-mRNA is alternatively spliced dependent upon the presence of Sxl protein
How does alternative splicing control sex determination in drosophilia?
- An early Sxl promoter is recognized and transcribed in female flies. Once zygotic transcription kicks in, both males and females start to transcribe Sxl gene.
- Sxl protein interacts with a specific region in the intron between exon 2 and exon 3 of the Sxl pre-mRNA being transcribed in both males and females during later development
- By interacting with that 3’ end, it blocks interaction with U2AF so cannot form cross-exon recognition complex in the intron-exon boundary
- When splicing takes place, exon 2 gets spliced to exon 4 because exon 3 doesn’t get recognized (Sxl is blocking it).
- Males try to transcribe the same thing and get the same pre-mRNA, but because they don’t have the early Sxl protein, there’s no interaction with that region in the 3’ end in the intron between exon 2 and exon 3. Therefore, males will splice exon 2 to exon 3 and exon 3 to exon 4.
- Once mRNA gets translated, there’s an in-frame stop encoded in exon 3, so they never make a functional Sxl protein
Sxl protein can go on to do more important things and bind to more RNAs in females
→ One of the most critical RNAs that it interacts with, with respect to sexual dimorphism, is the RNA made by the Tra gene (Tra = transformer).
→ Sxl will bind to 3’ end of intron meant to be excised and will block the formation that cross exon recognition complex and instead encourage the splicing complex to splice exon 1 to exon 3. In doing so, you end up forming a competent transformer (Tra) protein
→ Males cannot make a functional Tra protein (On exon 2, there’s an in-frame stop)
Once you make Tra protein, what does it do?
Once you make the Tra protein, it can interact with 2 other proteins: Rbp1 and Tra2 = both SR proteins
* Tra protein is going to bind to Rbp1 and Tra2 and decorate specific exons in females.
* They decorate Exon 4 to define to splicing machinery that this is an intron-exon boundary and the gene that they impact on is a gene called Double sex (Dsx)
* The splicing machinery knows that you have to include Exon 4 in this final product.
* By adding in Exon 4, there is also a polyadenylation signal that allows the formation of a Dsx protein made up of at least Exon 3 and 4 and a poly-A tail stabilizing it and making it useful to make protein.
* The male version of Dsx doesn’t have exon 4 and no SR proteins to decorate Exon 4 to tell the splicing machinery that it had to be included in a final mRNA product.
* In males, the splicing occurs between exon 3 and exon 5, exon 4 is skipped → mRNA will encode a protein with no exon 4
* Dsx is a transcription factor, and when you have a female version, a repertoire of genes typical to female diphormism is turned on In a male, a different male-specific sexual dimorphic repertoire is activated.
* End up differentiating male flies from female flies
Sexual-dimorphism is regulated by?
Sexual dimorphism is regulated by a post-transcriptional mechanism.
* An early Sxl promoter is recognized and transcribed in female flies
* Alternatively spliced variants lead to a functional TRA protein in “females”
* TRA/TRA2 and RBP1 specify a female specific doublesex gene product.
How does Sxl protein regulate splicing?
- Sxl binds to a sequence near the 3’ end of the intron between exons 2 and 3 and blocks the association between U2AF and the U2 snRNP (Thus Sxl represses a particular splice site).
- U1 snRNP binds properly to the 3’ end of exon 2, but assembles into a spliceosome with U2 snRNP bound to the branch point at the 3’ end of the intron between exons 3 and 4. Thus exon 2 gets spliced to exon 4 and exon 3 goes out as part of a larger intron.
Complex cascade of RNA binding proteins and regulation of the splicing apparatus → indicates if you will show female or male sexual characteristics.
- male embryos produce no Tra protein
- As a result of this cascade of regulated RNA processing depicted in Figure 9-18, different Dsx proteins are expressed in male and female embryos. The male Dsx protein is a transcriptional repressor that inhibits the expression of genes required for female development. Conversely, the female Dsx protein represses transcription of genes required for male development. In wild-type Drosophila, no Sxl protein is expressed in cells of male embryos, whereas it is expressed in female embryos. This is an example of “on-off switch” regulation. The initial expression of Sxl in early female embryos positively regulates its own expression. Other examples of nuclear RNA-binding proteins that generate switch-like control of alternative RNA splicing in neurons have been identified and termed master splicing factors.
Stability of mRNAs between organisms
The stability of cytoplasmic mRNAs varies widely within and between organisms
How can eukaryotic mRNAs be destrabilized
Eukaryotic mRNAs can be destabilized by a sequence motif
* Many short-lived mRNAs in eukaryotes contain multiple copies of the sequence AUUUA in their 3’ UTR
→ They are unstable or short-lived
→ They are important to confer the destabilization of the mRNA
→ If we put that sequence on a neutral mRNA, we can reduce the half-life to be similar to GMCSF
* Adding such sequence motifs to the 3’ UTR of a gene that usually does not contain them dramatically destabilizes the hybrid mRNA
Are all mRNA transcripts polyadenylated?
- Poly A tail stabilizes the mRNAs and protects them from exonucleolytic degradation
- All mRNAs are polyadenylated, with the exception of the histone mRNAs… they have unique secondary structure in their 3’ UTRs
- mRNAs that lack a poly A tail are rapidly degraded within the nucleus – this is part of the mRNA quality control machinery
- Although histone mRNAs (only exception) lack a poly A tail, they are not degraded. The histones have a stem loop structure at the 3’end that protects the transcript almost equivalently to a poly A tail.
Final Processing Steps required to make that pre mRNA into almost mature RNA
Polyadenylation of the 3’ end of the nascent transcript is required for stability.
* RNA polymerase II is released from the template at regions called the terminator.
* An AAUAAA xxxxx G/U sequence present within the 3’ region of the pre-mRNA is recognised by the cleavage and polyadenylation factors. This sequence contains the polyadenylation signal (AAUAAA).
* The 3’ end of the pre-mRNA is cleaved and then Poly A Polymerase (PAP) catalyses the formation of a poly A tail. The proteins that do this cleaving interact directly with the polyadenylation signal they include CPSF.
* The addition of the Poly A tail is necessary to protect the 3’ end of the product that you synthesized.
- The cleavage of a pre-mRNA molecule is coupled to the addition of a poly A tail
→ The moment that you cleave a pre-mRNA molecule, it becomes susceptible to any nucleolytic degradation from the 3’ end
→ Cleavage + protection in one event - Polyadenylation occurs in two phases:
→ A slow phase mediated by Poly A Polymerase (PAP) during which approximately 12 A residues are added on to the cleaved 3’ end
- Cleavage won’t take place unless PAP is present - you don’t want to cleave and expose the 3’ end until you are ready to protect it
- PAP will add a couple of nucleotides – it’s slow and lazy (approx 12 adenosine)
- When PABPII comes into the complex, it stimulates PAP - This structure is recognised by Poly A Binding Protein II (PABPII – in the nucleus – to distinguish it from the cytoplasmic PABP) which catalyses the rapid addition of ~200 A residues
→ Causes PAP to add nucleotides more rapidly
→ PABPII will then bind to the poly A tail
Binding protein or PABPN helps in adding in the poly A tail and makes polymerase much more efficient.
Poly A tail
3’ Polyadenylation
1. The 3’ end of the mRNA has a AAUAAA sequence. The Cleavage and Polyadenylation Specificity Factor (CPSF) binds to this site.
2. Three more proteins bind to the CPSF-RNA complex: [1] Cleavage Stimulatory Factor (CStF), [2] Cleavage Factor 1 (CF1), and [3] CFII
3. Poly(A) Polymerase (PAP) binds BEFORE the mRNA is cleaved
4. The pre-mRNA is cleaved at the Poly(A) site (downstream of the AAUAAA sequence)
5. The poly(A) Tail is added in two steps
A) PAP adds ~12 A residues slowly
B) PABPN1 binds to this short tail and quickly extends the tail by adding As for a total tail length of ~250 adenines
How can the stability of the RNAs be regulated
- Need transferrin to take iron in because it is toxic on its own
- The stability of the mammalian transferrin receptor TfR (which is needed for the import of iron into the cell) is regulated in response to intracellular iron concentration.
- Levels of transferrin receptor mRNA are controlled by playing or regulating its stability
- There are stem loops called IREs present in the 3’ UTR of the transferrin receptor mRNA
- They confer or destabilize the mRNA
- The IRE-BP that changes its conformation based on cellular levels of iron
- When iron levels drop below threshold, IRE-BP will change its conformation and will bind to the IREs
→ It protects the 3’ UTR of the transferrin receptor and allows for appropriate formation of the protein that will then confer entrance of iron into the cell - In the case of the transferrin receptor mRNA, it will bind to the stem loops which contain iron response elements, and in doing so, it will protect the mRNA from degradation
- During high iron, when the IRE-BP is in its non-binding conformation, the transferrin receptor mRNA is destabilized and it becomes degraded rapidly – largely due to stem loops that degrade it
RNA editing
In RNA editing (does not happen very often)…
* Single nucleotides are changed in the final mRNA and they are not encoded in the gene. The RNA’s are being modified during their maturation.
* mRNA is somehow recognized and specific nucleotides will be converted from one nucleotide to another
* The sequence of a pre-mRNA is altered. Therefore, the sequence of the mature mRNA differs from the exons encoding it in genomic DNA
→ RNA editing is widespread in the mitochondria and plastids of protozoans and plants
→ RNA editing is observed in the nuclear genomes of higher eukaryotes as well, however it is very uncommon
→ Deamination reactions remove amines to convert:
- Adenosine (A) to Inosine (I)
- Or Cytosine (C) to Uracil (U)
Example of RNA editing
Example: the mammalian apolipoprotein B
* Responsible for making bad cholesterol (LDL)
* Is involved in combining with other proteins to transport lipids around our bodies
* In the liver, the gene codes a large protein called Apo-B100
* In the intestine, a specific RNA editing takes place, converts a C to an U which will switch the CAA codon to a stop codon – makes the apolipoprotein B half the size of the Apo-B100
* Intestine has some specificity for altering the mRNA by changing one single nucleotide to make a stop that will end up forming a completely variant apolipoprotein so it corresponds only to the N-terminus of the full length Apo-B100 expressed in the liver
* Happens on the mRNA specifically – cell-type specific
