Fundamentals I: 11:00 - 12:00Scribe: Melissa Precise

Tuesday, September 15, 2009Proof: Matthew Davis

Dr. ChenPost-Transcriptional Regulation (Part I)Page 1 of 6

Post- Transcriptional Regulation [S1]:
If gives pancreatic RNAase just talking about RNA.
If gives enzyme, will tell if enzyme is exo or endo and whether it cleaves at A or B.
Knowing if the enzyme is endo or exo and where A and B is on the protein – you should be able to tell what kind of product you will get
A and B bullets from previous lecture.
Move on to Post transcriptional Regulation
Occurs between the transcription and translation
Material found in Chapter 29 but page numbers listed are probably incorrect
Crick’s 1958 View of “Central Dogma of Molecular Biology” [S2]
Have already had this diagram described
DNA can replicate by itself
Encodes from stored genetic information
DNA will not encode proteins directly so they use RNA to make proteins
Process to make RNA is transcription
Process from RNA to proteins is translation
Somewhere in between is a regulation to process this RNA which is called Post-Transcriptional Regulation
Principal Types of RNAs Produced in Cells [S3]
Does not think need to explain slide anymore.
Many kinds of RNA
Subject of our lecture is mRNA which encodes protein.
About 3-5% of total RNA is mRNA
What is Post-Transcriptional Regulation [S4]
What is post-transcriptional regulation?
Transcription is process to make RNA.
Either it is initiation, elongation, or termination
Process to make RNA is called transcription
So post-transcription is after transcription.
Occurs before transcription is fully complete.
Does not occur during translation, so at the beginning of translation there is no post transcription regulation occurring.
How are Eukaryotic Transcripts Processed and Delivered to the Ribosomes for Translation [S5]
There is a big difference between prokaryotes and eukaryotes.
Eukaryotes have nuclei while prokaryotes do not contain nuclei.
Regulation of gene transcription is different between eukaryotes and prokaryotes.
In prokaryote, transcription and translation are concomitant processes.
As soon as transcription is beginning, translation is already engaged because there is no barrier
In eukaryotes, transcription occurs in the nucleus and translation occurs in the cytoplasm.
Transcription of RNA needs to be transported into cytoplasm in order to be translated into the protein.
There is a difference in gene expression because of the structural difference between euks and proks.
On the way of transporting, processing occurs. This is today’s topic- processing steps that occur on mRNA. Purpose of this is to convert primary mRNA into mature mRNA.
Mature mRNA can be transported into cytoplasm and initiate translation
Prokaryotic mRNA can encode three proteins.
Eukaryotic mRNA is monocistronic because it can only encode one protein.
Eukaryotes vs. Prokaryotes [S6]
Because there is no nucleus in prokaryotes. RNA is transcribed.
There is no N terminal in prokaryotes.
No splicing occurs on prokaryotes.
As soon as RNA is transcribed, translation has already begun and no processing is undergone.
This is due transcription and translation occurring in the same compartment.
In eukaryotes, there is a nucleus and cytoplasm. DNA is stored in the nuclus and transcribed into primary mRNA.
It is not mature mRNA and cannot undergo translation into a protein. Can only undergo translation when mature.
Becomes mature in three steps of processing.
First is methylation and capping
Second is polyadenylation
Third is splicing
These three steps occur in the nucleus.
After the three steps, mRNA is mature and can be transported into cytoplasm for translation into the protein which is its main purpose.
Also eventually, mRNA needs to be degraded. If want to shut down expression of protein, then have to degrade mRNA.
mRNA can be localized or exported into some specialized part to initiate translation.
All of these steps are called post-transcriptional regulation which occurs between transcription and translation.
Comparison Between Prokaryotic mRNA and Eukaryotic mRNA [S7]
There is a structural difference between prokaryotic mRNA and eukaryotic.
5’ end and the 3’ end in the prokaryotic are not modified because the 5’ end contains phosphate and 3’ end contains an OH group.
One mRNA prokaryotic codes for three proteins which is called polycistronic.
There is a process that occurs in eukaryotic mRNA.
5’ end is capped with structure called 5’ end cap
3’ contains poly A sequence called Poly A tail
Euks encode one protein and are called monocistronic.
Processing of eukaryotic mRNA is subject of today’s lecture.
Eukaryotic Genes are Split Genes [S8]
Eukaryotic genes are called split genes because contain exons and introns.
Genes are split because not continuous due to containing the intron.
Introns intervene between the exons.
Exons are encoding regions and introns are noncoding regions (meaning they cannot encode the protein).
Usually exons are smaller than introns.
Exons are always first. Sequence goes Exon – Intron – Exon – Intron – Exon …
Give two examples
Actin gene
Relatively simple – contains 2 exons and 1 intron
Relatively small – only about 300 base pairs
Very tiny exon – only encodes 3 amino acids
So classified as a very simple gene
Chicken pro-alpha 2 collagen gene
If add together, about 40,000 base pairs
Contains 51 exons (exon – intron – exon)
Add all exons together – only about 5kb out of total 40kb
Rest of gene is introns – shows how big introns are
Exon site is very small – not more than 300 base pairs
Very tiny exons but huge introns.
Sometimes introns can be up to 10kb
Cannot translate into protein, so have to be removed
Remove introns by splicing.
This is complicated because intron is so big.
Splicing is very precise because if a mistake is made, then gene cannot be translated.
Intron needs to be removed in the precise position.
Eukaryotic Genes are Split Genes (Figure 29.36) [S9]
Cartoon of how eukaryotic gene looks like.
Have DNA. Need promoter to initiate transcription. RNA polymerase transcribes from gene to form primary mRNA which contains introns and exons.
Introns have to be spliced out to start forming mature mRNA to be ready for translation.
The 5’ cap and 3’ Poly A tail is added.
Bottom figure shows mature mRNA which is ready for translation. Notice it only contains exons, the 5’ cap, and the 3’ Poly A tail.
The Organization of the Mammalian Dihydrofolate Reductase (DHFR) Gene [S10]
Shows similarities of one gene from three different mammalian species of the mammalian dihydrogolate reductase (DHFR) gene. Same gene from the difference species (Chinese hamster, Mouse, and Human).
When compare side to side, can tell the exons are very converse while the introns are very diverse.
The gene is split into 6 exons and spread over 31kb.
Whenever the 6 exons are spliced together for the three mammals, will all give a 6kb mRNA.
Shows how the exon pattern is more highly conserved than the intron pattern.
mRNA Processing Involves [S11]
Three events occur on mRNA in the nucleus.
The processing of mRNA includes capping and methylation, polyadenylation, and splicing.
Will swap splicing and polyadenylation in order to describe splicing last.
Capping and Methylation [S12]
First is capping and methylation.
As soon as primary mRNA is synthesized by RNA Polymerase II (about 10 nucleotides), the 5’ end is already capped (occurring before transcription is complete).
The capping is catalyzed by guanylyl transferase and uses GTP as a substrate to add Guanine (G) to the 5’ end of Cap 0.
Capped G is always methylated at the N7 position.
The Capping of Eukaryotic pre-mRNAs [S13]
Guanylyl transferase catalyzes the addition of a guanylyl residue.
So capped structure is always GTP.
Figure 29.38 Methylation of Several Specific Sites [S14]
Additional methylation will add to the N7 position which occurs at the first or second nucleotide by another enzyme at the C2’-O positions of the next two residues at 6 amino groups of the first adenine.
Will see slide later showing this.
The additional methylation on the first nucleotide is called Cap 1 and the methylation on the second nucleotide is called Cap 2.
90% of mRNA contains methylation on the first nucleotide called Cap 1.
Don’t have to remember Cap 1 and Cap 2 – will not ask a question on these.
Cap structure contains methyl group. Structure of 5’ end contains Cap 0.
Enzymes Involved in the Capping [S15]
Three enzymes are involved in the capping.
First enzyme is phophotase
Removes phosphate group at first nucleotide
Second enzyme is guanylyl transferase
Adds the GTP to the 5’ end of the cap structure
Third enzyme is guanine 7-methyl transferase
Transfers methyl group to Cap G
Why Do Cells Need to Cap Their mRNA? [S16]
Several reasons cells cap their mRNA.
The cap is recognized by protein complex called cap-binding proteins.
Cap distinguishes mRNAs from other types of RNA molecules
Three types of polymerases can be found in the cell.
Only polymerase II can be used to make mRNA.
So when see a cap structure on the mRNA, know that mRNA was made from polymerase II
mRNA needs a cap and will talk later about poly A tail which is also needed to export mRNA out of the nucleus.
Cap is also necessary for translation.
Cap can stimulate translation with the protein bound to the Cap. Cap protects the mRNA from degradation by stabilizing it in the cytoplasm.
RNA Factory – mRNA Processing is Coupled to Transcription [S17]
Another feature about gene regulation.
Post-transcriptional regulation is coupled to transcription. It is not separated.
Somehow transcription can regulate the processing of mRNA.
There is a C-terminal domain called CTD of the largest subunit of RNA polymerase II.
CTD contains 52 copies of heptapeptide (7 amino acids)
This can be phosphorylated by kinase during elongation which provides a binding site for factors involved in capping, splicing, and 3’ end formation.
Enzyme already there because tied with CTD. This is why capping on mRNA occurs when nucleotides have been transcribed.
Capping is coupled to transcription.
If have a mutation on C terminal domain, then no longer associated with capping enzyme which will not allow capping to occur. Shows the coupling between transcription and post-transcriptional regulation.
3’ Polyadenylation and Transcription Termination [S18]
Second event is polyadenylation.
Jump to 3’ end now.
Somewhere at the 3’ end of the gene, RNA Polymerase II transcribes mRNA.
Somewhere at the 3’ end, the mRNA will be cleaved in order to be removed from DNA.
After the mRNA is cleaved, the poly-A tail will be added to the cleaved 3’ end.
Focus is on the sequence and also the protein end in the 3’ polyadenylation.
3’-Polyadenylation [S19]
Somewhere, somehow – the 3’ end will be cleaved and transcription will end. Will stop making mRNA.
Want to shut off making mRNA. This termination requires a consensus sequence.
Consensus sequence is AAUAAA. This is the poly-A signal.
RNA polymerase II transcribes up to this part. When sees this sequence will terminate.
About 10-30 nucleotides downstream, the mRNA is cleaved.
Once cleaved, about 200 adenine residues will be added to the 3’ end of mRNA known as the poly A tail.
There is another enzyme that can add Adenine residues to the 3’ end which is called Poly A polymerase.
Poly A tail by recognition of Poly A binding protein protects from degradation and stimulates translation as well as governs the stability of mRNA.
Signals Required for the Formation of the 3’ End of mRNA [S20]
Two signals are required for specification of the 3’ end of the gene.
First signal is AAUAAA.
Second signal is downstream and is GU rich.
About 30 nucleotides between first and second signal, mRNA will be cleaved.
The poly A tail will then be added by poly-A polymerase specific to the 3’ end.
These two signals are specific to the 3’ end.
Mammalian pre-mRNA 3’ End Processing Complex [S21]
Do not have to memorize this.
This sequence is recognized by a protein complex. The protein complex is recognized by poly A signal called CPSF (Cleave Polyadenylation Specificity Factor).
CPSF contains four proteins
There is another protein complex that contains three proteins called CstF (Cleavage Stimulation Factor). This recognizes the downstream GU-rich sequences.
These two protein complexes come together to form a protein-protein interaction.
There is an enzyme that can cleave mRNA and another enzyme that adds the poly-A tail.
This is how cleavage of mRNA and addition of poly-A tail occurs.
Figure 20.39 [S22]
This is a simplified cartoon to show how a 3’ end is recognized.
Here is RNA polymerase II transcribing mRNA which recognizes the poly-A sequence.
Now the protein complexes interact with each other.
The complex adds the poly-A tail after the 3’ end is cleaved.
Usually about 200 Adenine residues are added at the 3’ end.
Polyadenylation is Coupled to Transcription [S23]
Similar to capping, polyadenylation is coupled to transcription.
Again, C Terminal Domain is preferentially phosphorylated and intact with cleavage polyadenylation specificity factor or cleaage stimulation factor.
C Terminal Domain associate factor is present as soon as poly A factor is transcribed.
If mutate C terminal domain, polyadenylation will not occur.
Polyadenylation of mRNA [S24]
Do you find a poly-A tail in your gene? Do you find 200 adenines in your gene?
Because adenine is added after the mRNA is cleaved, you will not find this in your gene.
So do not need a template for the poly-A tail.
Transcription needs a DNA template, but poly-A tail does not need template because it is added.
Poly-A tail is recognized by poly-A protein which is needed for translation as well as protection of mRNA from mRNA degradation.
Pre-mRNA Splicing [S25]
Already have capping and polyadenylation, now move onto splicing.
What is splicing?
This is a simplified cartoon.
This is a very simple gene because it has 3 exons and 2 introns.
Transcribed into mRNA and spliced into mature mRNA.
Once spliced, the mRNA is considered to be mature and can be transported into cytoplasm which then allows translation of protein.
Nuclear Pre-mRNA Splicing [S26]
Splicing only occurs in the nucleus.
Primary mRNA or pre-mRNA is recognized by the protein binding protein from RNA protein complex.
We call this pre-mRNA association to the RNA binding comples –RNP or ribonucleoprotein particles
Want to assemble the RNP in manageable order or structure. Primary structure can be 40kb, so very long.
Need to assemble proteins onto this pre-mrNA from RNA protein complex to make the mRNA smaller.
Makes the complex able to manage the processing.
Pre-mRNA is always associated with RNA binding protein.
Substrate for splicing comes from RNA Polymerase II which are intron excision and exon ligation.
Splicing occurs in the nucleus.
Can align introns from different genes together and will have consensus sequences between each other.
Splicing of Pre-mRNA [S27]
Primary mRNA is capped and polyadenylated RNA in the form of a RNP complex which is the substrate for splicing.
Splicing is where excise introns and leave the exons together.
The 5’ end of an intron is always GU and the 3’ end is always AG.
All introns have a branch site 18 to 40 nucleotides upstream from 3’ splice site.
Branch site is essential to splicing.
What Makes an Intron? [S28]
Essentially an intron.
Upstream exon and downstream intron.
Align intron from different genes- have consensus sequence.
GU sequence found 5’ end and find AG sequence at 3’ site.
Only the A in the branch site is never changed.
These are three things required for splicing.
If have mutation on any of the three sites, splicing will not occur.
The Splicing Reaction Proceeds via Formation of a Lariat Intermediate [S29]
Chemical reaction of splicing is the formation of Lariat intermediate.
Will show how Lariat intermediate looks later.
Lariat intermediate is formed by a 5’ invariant G (remember 5’ end is always G and U).
When form a Lariat intermediate, the 5’ end G will link to the A in the branch site
Forming the intermediate is the first step.
The second reaction excises the lariat intermediate. This joins the 5’ exon with the 3’ exon.
Lariat intermediate is unstable and will be quickly degraded.
This is the simplified chemical reaction.
The Splicing Reaction Proceeds via Formation of a Lariat Intermediate (Pictorial) [S30]
5’ end is always GU and branch site is always A.
1st reaction – G forms covalent bond to A site.
Now cleave the exon/intron junction and expose 3’ end of previous exon and now the second reaction will cleave after the 3’ end of intron.
Once excise Lariat intermediate, the two exons will join.
Intron is excised and will then be degraded.
Very simplified chemical reaction.
Splicing Depends on snRNPs [S31]
The protein required for splicing is a set of small nuclear ribonucleoprotein particles known as snRNPs.
Pronounced “snurps”
Each snRNP consists of one small RNA and about 10 proteins.
Some are the same proteins, but some proteins in snRNPs are more specific (or different for that specific mRNA).
Will tell how many snRNPs there are, later.
snRNPs are very abundant.
snRNPs and pre-mRNA form spliceosome.
Will tell what spliceosome is later.
Splicing Depends on snRNPs (Table) [S32]
There are five difference snRNPs which are required for splicing – U1 U2 U4 U5 and U6.
RNA found in snRNPs is very small. Only one RNA per snRNPs.
snRNPs are associated with 10 different proteins.
Contains small number of nucleotides which can be seen from the table.
U1 recognizes 5’ splice
U2 recognizes branch site
U4, U5, and U6 always come together (associate together).
If intron is very far away, say 10kb, then how can 5’ end recognize branch site. Use 3 part snRNP to bring closer together so can react and form the intermediate.
snRNPs Form the Spliceosome [S33]
snRNPs form the spliceosome.
Spliceosome is the machinery containing the primary mRNA and snRNP – forms the very large multi-component complex known as the splicesome. The spliceosome is about the size of a ribosome.
U1 recognizes 5’ site and U2 recognizes branch site.
Interaction between snRNPs brings the 5’ and 3’ end together so Lariat can form and exon ligation can occur. Spliceosome requires ATP and also requires other proteins – not only snRNPs.
The Spliceosome – RNA/Protein Complex [S34]
Spliceosome contains mRNA, snRNPs, and other proteins to form the huge complex that catalyzes splicing.
RNA component contains five components (U1, U2, U4, U5, U6).
The 5’ splice site and branch site are recognizes with the help of other proteins called non-snRNP splicing factors.
These are SR family proteins and other splicing factors.
If ask how many total proteins are found in the spliceosome – would be estimated to be more than 50 different proteins.
snRNPs Form the Spliceosome [S35]
Here is shown how U1 assembles onto 5’ splice site.
Forms a secondary structure with protein where its 5’ end is single stranded. This structure recognizes the 5’ splice site.
Now have the first step in splicesome assembly – which is recognizing the 5’ splice site.
Next step is recognizing branch site.
Assembly of the Spliceosome [S36]
Summary of previous slides.
Figure 29.44 Events in Spliceosome Assembly [S37]
Very complicated slide. Do not have to remember.
Two things to remember:
First: The 5’ site
Second: The Branch site
Then U4, U5, and U6 always comes in together to assemble the machinery.
First chemical reaction occurs for the Lariet intermediate to form, then protein RNA rearrangement occurs.
Brings 2 ribosomes together and second reaction occurs.
In the second reaction, the RNA is excised bringing the two exons together. snRNPs are recycled for initiation of next splicing reaction.
Pre-mRNA Splicing is Coupled to Transcription [S38]
Splicing also coupled to transcription.
Want snRNP to associate with C terminal domain (RNA Polymerase II).
As soon as mRNA is transcribed, then snRNPs can attach.
If interfere with transcription, then also interfere with splicing.

[end 51 min]