Flashcards in Transcription II RNA Processing Deck (21):
How did we start this lecture
We started the lecture by talking about how these mutation stories go with disease development. He basically talked about how these lectures are setting us up for genetics and neoplasia.
In the first slide it said that single gene mutation, null mutations results in rare congenital defects that results in devastating outcomes usually.
Furthermore, allelic variations at single or multiple locations or loci can also lead to contribution to risk factor for a disease.
When does mRNA processing occur
The next slide said that mechanistic insights of null mutations can give us information about the modest changes in protein structure and expression of normal allelic variants.
Although we tend to think of these events as sequential, occurring after RNA synthesis is complete, in fact they occur during RNA synthesis. The C-terminal domain of RNA polymerase II binds to and organizes the enzymes necessary for capping, splicing and polyadenylation.
What are the 3 rNA processing
Today we talked about RNA processing. That involves, capping, slicing and polyadenylation
The three RNA processing are:
1. Capping (occurs co transcriptionally)
2. Cleavage and polyadneylation (this happens at the 3' end and forms the 3' end)
3. Splicing (occurs co tranriptionally)
Unique thing about the RNA processing of homo sapiens
He said we have only a few part of the DNA that encodes for genes, like corn has more genes than us. But we are complex organisms and this complexity arises from the fact that we can process RNAs differently to produce a variety of proteins.
Then we dived into RNA capping: it is a 7-mthyl guanosine cap.
1. Occurs shortly after transcription
2. Guanlyltransferase is the enzyme involved. It adds G group to the 5' end.
3. Cap has a 5' to 5' triphosphate bond (know how this looks like, you need special exonucleases to break this, their called decapping enzymes)
4. Methyltransferases add methyl groups (know where these methyl groups are, it is on 2 nucleotides)
a. To the 5' terminal
b. At the ribose of 2 OH on next 2 nucloetides
Functions of capping
Function of capping:
1. Increase mRNA stability
2. Can be exported to cytoplasm
3. Required for translation
When does cleavage happens
Then we talked about cleavage and polyadenylation. Cleavage happens after a specific sequence.
Polyadenylation happens, it is catalyzed by poly(A) polymerase. This involves 150 to 200 A residues being added at the end, this does not requires a DNA template
Role of adenylation
It said on the slide that polyadenylation marks the mRNA as special
1. It has a role in translation efficiency
2. Exported to cytoplasm
3. Stability of mRNA
What is the key for proteome diversity
Now we talk about splicing. This is very important. This is the key for proteome diversity that is found in homo sapiens. This slide basically talked about how mRNAs of different genes may vary in lengths by either having no intorns, some introns or many many introns. Following are his comments
This figure shows examples of the wide variety of gene structures seen in the human genome. Some (very few) genes do not have introns. One example is the histone genes, which encode the small DNA-binding proteins, histones H1, H2A, H2B, H3, and H4. Shown here is a histone gene that is only 400 base pairs (bp) in length and is composed of only one exon. The beta-globin gene has three exons and two introns. The hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT) gene has nine exons and is over 100-times larger than the histone genes, yet has an mRNA that is only about 3-times larger than the histone mRNA (total exon length is 1,263 bp). This is due to the fact that introns can be very long, while exons are usually relatively short. An extreme example of this is the factor VIII gene which has numerous exons (the blue boxes and blue vertical lines).
How does complexity and diversity in human proteome arise
Complexity and diversity arises from the utilization of both alternative splicing and termination/polyadenylation. One mRNA can give rise to many different proteins. It is important to know here that where ever the polyadenylation sequence is added in the mRNA then that is where the mRNA will be spliced off temrinally (it will end). Remember the diagram that he showed us in the lecture where there were polyA sequences added at different intervals of the exons in mRNA which gave rise to different lengths of the protein from the same mRNA.
Details of the first case study of this lecture
We then did a case study. A baby of parents who were cousins was found to be underweight and presented with a chief complaint of inactivity and poor duckling, decline began at day 30 after birth. The baby looked alert and has normal cranial nerve function. Family history is negative for neuromuscular disorders.
He had a hypotonic phenotype called the floppy baby phenotype. Upper extremity power is low. Serum creatinine kinase was found to be elevated in the blood, this is a marker of muscle damage.
Additional tests showed lower than normal nerve conduction velocities.
At 4 months the baby could not breathe on its own and had to be put on a ventilator.
Finally genetic testing confirmed spinal muscular dystrophy.
He showed us a video of a child who had symptoms of spinal muscular dystrophy.
SMA is a primarily motor neuron degenerative disease. Sensory neurons loss is sometimes seen. There is significant physical and developmental defects and there are several forms of this disease depending on the mutation itself.
The disease is autosomal recessive, loss of survival motor neuron 1 gene expression, SMN1 gene
8 million people in the USA are affected by this disease
Details about the biochemical mechanism of SMD
snRNP are chaperons and catalyze splicing
SMN1 gene makes SMN1 protein, this interacts with snRNP (small nuclear ribonucleoprotein) . SMN1 protein is needed for snRNP assembly.
A similar gene called SMN2 can partially compensate for the loss of SMN1. (the mRNAs lack exon 7 in SMN2 as compared to SMN1).
Microanalysis of SMN1 knockout revealed that there are widespread pre-mRNA splicing defects leading to hundreds of diverse transcripts that are affected by this mutation, mRNAs from genes with large number of introns are especially affected.
The end result is that motor neurons start dying.
The next slide said that mRNA content is qualitatively and quantitatively reduced altered due to reduced snRNPs.
How does snRNPs know where to do splicing
Now we look into the role of snRNPs.
Splicing signals are conserved nucleotide sequences.
He showed an example of splice sequence. Not sure what was that about. Mammalian splice signals are specific and conserved.
snRNPs recognize these introns and exons boundaries and carry out the splicing as directed by these sequences. Loose splicing sequences leads to null mutations.
How does splicing happen biochemically
If there is a mutation in the splice signal, there is a loss of splicing.
Splicing signals often mutated in genes are inactivated that leads to disease. (it had a comment on see the end of lecture to read about beta thalessemia)
Then we talked about how does splicing happens biochemically.
Splicing intermediate is called the Lariat structure. There are 2 SN2 type reactions:
1. 2' OH on adenosine at branch point (or slice point) attacks the 5' G of intron. The inton's end as a result winds up on itself.
2. OH-G at 3' end (to the left) of the exon attakcs the 5' G of the next exon in sequence (at the 5' end, to the right).
As a result, the intron winds up on itself and the two exons combine, having a phosphate group in the center.
Don’t be too hung up on this chemistry
More details about snRNPs, types of snRNPs and a disease associated with snRNPs.
We then go back to the topic of snRNPs. He mentioned Protein-RNA-mRNA complex as the splicing machine. These are small nuclear RNAs (or snRNAs), snRNAs associate with proteins. Loss of SMN1 leads to a defect in the assembly of these snRNAs.
RNA + snRNPs = snRNA.
snRNPs hold the mRNA in place and calatalyses the reaction. There are different kinds of snRNPs or snRNAs. snRNAs are U1, U2, U4, U5, U6 (no U3).
snRNPs forces pre-mRNA into arrangement that enables activation of RNA catalytic center that is responsible for intron removal.
He then mentioned something about antibodies directed to snRNPs leading to development of systemic lupus erythematosus (SLE is an auto immune disease).
These snRNPs are present only in eukaryotes.
Further details about the mechanism of each snRNPs and what did Max lacked that lead to his disease
Then we talked about the mechanism of the snRNPs action or mechanism of spliceosome.
U1 and U2 recognizes the key sites where splicing needs to occur (from the 5' for U1 and 3' end for U2). He said something about U1 is at the donor exon/intron boundary and U2 is at the adenosine at the branch point.
U4,5 and 6 come in to form a spliceosome. Then it had a comment that alltogether these snRNPs:
1. Ensure proper geometry for splicing
2. Hold together exons prior to joining
3. Catalyze cleavage and formation of bonds
Then in the next slide we talked about the fact that Max doesn’t make enough of these snRNPs so how does that cause his disease (what is the correlation)?
1. Insifficient numbers of snRNPs
2. Inefficient splicing
3. Proteins misisng, no fucntion, motor neurons are particularly sensitive
4. Normal ratio of splice variants is altered
Different kinds of exon processing
As an example he showed us patterns of alternate exon usage which basically means different ways we can process mRNA to make proteins that are different but belong to the same class of proteins.
The slide said that one gene can produce several different but related protein species (which are isoforms). The following are the examples of exon processing:
1. Cassette skipping (skipping exons)
2. Mutually exclusive
3. Internal acceptor site
4. Alternate promoters
Broader significance of splicing in human genome
Then we talked about broader significance of mRNA splicing in Human Health.
More than 90% of the human genes are alternatively spliced. These are required for gene regulation during development, cell differentiation and homeostasis.
- snRNPs leads to assembly defect leads to SM atrophy or SMD
- Mutations in a single nucleotide of splice acceptir or donor site can result in functionally null mutation of a gene
- SNPs in elemetns that contribute to regulation of splicing can alter the function/disease risk
This is a source of evolutionary material: Exon shuffling among genes.
Then he had a comment which said that a growing evidence suggests mutations in splicing factor expressions leads to cancer development, progression and response to therapy.
What was the 2nd case study about
This case study was used as a starting point to talk about a risk factor KLF6. Its called kruppel like factor. Binds with DNA and regulates transcription, its an anti cancer protein. There is a variant that is shorter, it is dominant negative as it suppresses the activity of regular KLF6.
Wild-type KLF6 typically induces cell cycle arrest, including via the induction of CDKN1A, and opposes epithelial–mesenchymal transition (EMT) by activating CDH1 (which encodes E-cadherin), whereas KLF6-SV1 suppresses CDKN1A and CDH1 to enhance tumor progression
He then showed us a graph of the ratio of mutated over wild type KLF6. In metastatic cancer this ratio is larger, the larger the ratio the less likelihood of survival of the patient.
It is a useful biomarker.
Now we talk about the mechanism of KLF6.
This is an example of single nucleotide polymorphism (SNP) which is assocaited with prostate cancer.
The mechanism basically is that protein binds with higher affinity with the DNA for the mutated KLF6.
SNP in splicing
In the past we have talked about single nucleotide polymorphism SNPs that affect transcription, here we talk about how SNPs affect splicing.
He then just showed us 2 slides on how alternate splicing is used to make essential proteins in a cell. Here we talked about the example of glucocorticoid steroid receptor (remember how the enhancer and suppressor are made, they differ by the last 2 exon, active have the first exon, suppressor have the 2 exon (skips the 1st exon at the end).