Lecture #9 - RNA Processing Flashcards
Use of RNA
Overall - RNA is key to protein expression and gene expression
- Messenger that brings information from DNA to protein
- Important macheinery that tranlsates RNA to protein (rRNA)
- Needed to make RNA to mRNA (uses snRNA)
- RNA is needed to make tRNA
Is transcription the only thing that is needed to make RNA
NO RNA is ONLY made by transcription
Transcription is the begining of the production of RNA BUT after transcription RNAs need to be processed to mature forms that are used for gene expression
- There are many enzymes that transform RNA molecules
Types of RNA species used in the central dogma
PiRNA and siRNA = involoved in protein regularion
Where do things happen in Cells
Eukaryotes:
- Nulcues - DNA replication + transcriptional + RNA processing + RNA decay
- Cytoplasm - RNA processing + Tranlsation + RNA decay
Bacteria:
- Cytoplasm - DNA rpelication and transcription + RNA posicessing + tranlsation + RNA decay
When does RNA proccesing often occur
RNA porcessing = often occurs co-transcriptionally
Eukryotic RNA polymerases
Eukaryotes have many RNA polymerases that transcribe different RNA products
- Type of RNA Pol that makes the RNA determines the dwonstream processing effects that RNA will go through
Types of RNA Pol:
Pol 1 = makes rRNA
Pol 2 = makes mRNA + snRNAs + snoRNAs + miRNAs
Pol 3 = makes tRNA + 5S rRNA + other small RNAs + U6 (splisomal RNA)
*** Bacteria = have 1 RNA pol
Things that can occur to transcripts
Things can be done to RNA as it goes from primary transcript to mature RNA
- Most RNAs require multiple processing steps
Things that can happen to RNA to make the mature form:
1. Cleavage
- To have presice ends on the RNA prodict you need to cleave the ends with enodnuclease or exonuclease
2. Splicing
3. terminal nucleotide addition
4. Editting (Insertions + Deletions + Modifications)
- Editting = changing the nucleotide sequence in the RNA sequence
rRNA modifications
- Multiple cleavge events
- Nucleotide modifications
Note - rRNA transcription + procesisng + assembly are ALL coupled
Ribosomes
Function - Translate mRNA to proteins
Ribsome = made up of RRNA and proteins (mostly made of rRNA)
- Ribosome = has large SU and a small SU
rRNA transcripts
rRNA = made from polycystronic transcripts
- rRNA will make up the ribosome once they are processed
- Nascant RNA will be processed and assmbled o form mature ribsoomes (rRNA will start to fold and asscoiate with teh correct proteins to give a precsise structure)
Image – 3 rRNA in 1 transcript (3 blue parts)
- Cut out the 3 parts to make mature rRNA from the larger RNA moelcule
Why make polycytronic transcripts from rRNA
Ensures that the different rRNA is made in the same stocihemtriric amount
RNA pol 1
RNA polymerase 1 = strong polymerase –> intiates transcription and transcribes RNA very efficinetley
rRNA genes
rRNA genes – 10-44 kb regions (repeated throughout the genome)
rRNA genes = make polycyctronic RNAs = ensures equimolar amounst of the various rRNAs
rRNA coding sequences in gene = accounts for 1% of the genome BUT generates 40% of the trancriptional outpyt f the cell
rRNA transcription in a cell
Can see rRNA transcription in a cell –
When you spread the region of the chromosome that contains rRNA repeats you can see rRNA emerging from transcripts
- Can see how many transcripts are being made from 1 cystron
- ALSO can see forms balls where the ribsomes are being assembled
Making mature Ribosomes
- Cleavge
- Folding
Making mature Ribosomes (Cleavage)
As rRNA transcript is transcribed it will be cleaved (need to cut out the rRNA from the precursor)
- Enzymes used = endonucleases + exonucleases
- Endonucleases and exonuleases release rRNAs
Ends that the endouclease produces are NOT precsies –> AFTER need trimming by an exonuclease
- Ends are primiaity defined by endonucleases but exonucleases can also refine them
USES RNASe 3
- Precise ends of mature mRNA and tRNAs and most RNAs are genrated by clevage by RNAses
RNAse 3
Overall - Processed rRNA and other RNAs
Function - cleaves dsRNA and intramolecular stems (ex. Pre rRNA, snoRNAs, snRNAs)
- Mg2+ dependnt endonuelease
Process - RNAse 3 = recognize dsRNA that forms a stem loop –> RNAse 3 makes a staggard cut (Leaves a 2 nucleotide 3’ overhamge
- Generates ends with a 5’ PO4 and 3’OH
Making mature Ribosomes (Folding)
Location - Most assmley of the ribsome happens in the nuceleolus BUT some maturation continues in the neoplasm
- Have co-transcrtional dynamic association with processing factors as the rRNAs fold and binds to ribosomal proteins
Partciles that are exported from the nucleus will continue maturing in the cytoplasm
- Can have exchnage of proteins in cytoplasm
tRNA modifications (Overall)
- Cleavge
- Splicing
- Terminal nucelotide addition
- base modifications
TRNA makes larger precursor –> precusor folds into clover structure
Processing tRNA (Cleavage)
Precursors are longer than the mature form
- Precursors have a 5’ leader and 3’ trail –> need enzymes that will cleave at specifc positions
RNAse P = removes 5’ leader
RNAse Z = removes trailer
BOTH RNAse P and RNAse Z = only care about structure NOT sequence
- Many RNAses rely on ructure for specificty
- RNAse P and Z = endonucleases
RNAse P
Function - removes 5’ leader of tRNA
Made up of mosty RNA BUT some proteins
- Number of proteins increase form bacterua (few proteins) –> archea –> Euk (many proteins) BUT Mechansim of enzyme is conserved
How does RNAse P recognize the tRNA:
In 3D fold tRNA is in L structure –> RNAse P recognizes the tRNA by measuring the length of the horizontal arm on tRNA –> RNAse P can find the end of the tRNA and cut out the lead
Application of RNAse P and RNAse Z
Can use RNAse P and Z to cut out guide RNAs for multiplex CRIPSR editting
- Can express multiple guide RNAs for CRIPSR
Often = express gRNA under a Pol 3 promoter BUT that can only put 1 gRNA
INSTEAD – can put gRNA between tRNA and express this in transcript –> cells recognize tRNA and will cut precisely between the tRNA = release the long transcribed gRNA for multiplex CRIPSR
Processing tRNA (Splicing)
Some tRNA have introns that need to be removed by cutting and splicing ends together –> THEN need to join ends using tRNA ligase
- Done using the TSEN complex (TSEN = type of endonuclease)
Processing tRNA (Terminal Nucleotide addition)
Overall - Add 3 nucleotides at the end of tRNA
- ALL tRNA have a CCA at the 3’ end
- CCA = pairs with rRNA to position the tRNA correctly for peptide bind formation
Processing tRNA (Nucleotide modification)
Many rRNAs and tRNAs have many modified nucleotides + Happens at many positions in rRNA and tRNA
- Up to 25% of nucleotides are modified in tRNA
Ribonicleotides can be modified in >100 ways
Modifications can happen at all possessions on bases
Some modifications affect W/C face = affects the BP capacity BUT some can be modified on the other side and affect the way RNA folds
Type of nucletide modifications
- Methylation
- Isomerization of U
- Demaination (Ex. demiantino of adenosine makes an Inosine)
- Remove the Amino group that is used to BP A-U
- Insoine = behaves like a G and pairs with a C
- Psudeouracil
- Made by cutting the glycosidic bond –> rotates the base
- Modify the surgar – methylate the 2’OH on the ribsone sugar
Consequences of nucleotode modifications
Nucleotide modifications affect stricture and the ability to interact with other RNAs and protein
- Modulationes of base pairing and decoding
- Structure stabilization
- Bidning to specific readers
- 2’ OH methylarion affescts sturcture and stability
Location of tRNA nucleotide modifications
Modification occurs throughout the tRNA body AND have some within or next to the anticodon
Anticodon nucleotides
tRNA uses nucleotides at positions 34,35,36 for anticodon (Forms basepairs with the nucleotides in codon of the mRNA)
- Bases positioned at 34 = tend to be modified
tRNA reading codons
tRNA is able to read non-perfect codons (have wobble position in the 3rd position)
- Modified base in position 34 = can read the wobble position imprecisely = enables the tRNA to read multiple codons
- Can have different nucleotides in the 3rd position
- Position 34 of the anticodon tRNA can have whole sugars added onto the base
- Position 37 (NOT in the anticodon) = modified so that it fixes the position of the codon/anticodon interactions
Consequences of different types of modifications on tRNA
- Have modifications that affect RNA structure (important for tRNA)
- Have modifications that can be recognized by proteins – provides specificity for readers that recognize RNA
- 2’OH methylation = provides stability
- RNA is unstable because 2’ OH is a nucleophile that can attach the phosphodietsrer bond of the adjacent base and cleave the RNA molecule from within BUT if the 2’OH is methylated then it can’t do that
How do enzymes know which nucleotide to modify in rRNA
Overall - Specificity of modifications = sequence based + affected by structure (because regions need to be single stranded)
Specificity of rRNA modifications is determined by base pairing interactions with snoRNAs that guide enzymes
- Most modifications are added as the RNA is being folded
- Modifications are guided by snoRNPs
Most common modification on rRNA
Most common modifcation on rRNA = 2’OH methylation and Pseudouracil
snoRNPs
SnoRNAps = SnoRNAs + proteins
- SnoRNAs = enzyme that preforms the modification
- RNA compenent = gudies the enzymes by BP complenetary of 2 RNAs (Similar to cas9 finding the target using a gRNA )
Example 1 – fibulin us guided by snoRNA (BOXC and BOXD snoRNA)
- BOX D and BOXC = BP with RNA and the enzyme will methylate positions within the base pairing
Example 2 – Box H and Box ACA react with dyskerin/CbfP5 to make uradin to psuedouradin
How do enzymes know which nucleotide to modify in tRNA
Overall – Specificity of tRNA modifications relies on global and local structures
- tRNA modifying enzymes use structure and sequence for specificity
Examples:
1. Pseduradine Syntehases (PUS)
2. Methytransferases (TRMT, NSUN, METTL)
3. Adenosine Demainase (ADAR)
Pre-mRNA capping Process
Start - RNA polymerase begins transcription with a nucleotide tripospahte (ex start with GTP – have PPP at the 5’ end of RNA product)
- 5’ phopshate (gamma phosphate) = removed and a GMP is added to the remaining diphosphate (Step 2)
- WHEN added the GP is added in a different link form –> creates a 5’-5’ linkage (5’-5’ linkages = prevents degradation by exonuclease)
- Cap = protects the 5’ end of mRNA
- WHEN added the GP is added in a different link form –> creates a 5’-5’ linkage (5’-5’ linkages = prevents degradation by exonuclease)
- Methylation of added to position 7 of guanine
CPSF
Used for 3’ cleavage and Polyadenylation
- System for polyadenylation = has recogintion and then stimulation
CPSF = comes with RNA pol –> when RNA polymerase transcribes the AAUAAA sequence CPSF can bind BUT it can’t do anything until Pol transcribes the GU rich sequence
THEN when have transcription of the GU rich sequence the CstF (cleave stimulation factor) binds = activates CPSF for cleavage –> Cleave occurs at CA dinucletides THEN PABP comes and adds a poly A tail tp the 3’ end
Use of PolA tail
PolyA tail – used for mRNA enrichmemt
- Use polA tail when want to purify just mRNA by pulling down on polyA mRNA using beads or using a oligioDT primer that will hybridize to polA
NOTE - ONLY 3% of cellular RNA is mRNA (40% is rRNA) = needs to be enriched for = need to pull down and isolate it
Exception of 3’ polyA tail
EXCEPTION of 3’ polyA tail = histones mRNA which lack polyA tail BUT have stem loop structure at the end bound by SLBP
Stem loop at 3’ end = generated by U7 snRNP-mediated reaction
- U7 snRNA cleaves at position and allow for bidning of SLBP
- SLBP = holds onto the stem of the histone mRNA and protects it during tranlsation
Discovery of Splicing
Found cells infected with adenovirus –> purified the genome of the adenovirus and purified the adenovirus mRNA by taking the mRA that was being translated by the ribosomes
THEN allowed the DNA genome and the mRNA of the virus to hybridize –> looked at electron microscope
In microscope = saw RNA hybrdized to one part of the genome THEN skipped a part then continues hybridizing further downstream
***Shows mRNA is not fully contiguous with the genome
What makes an intron
There are concensus sequences that define the end of the exon and the begining of the next one (the intervening intron seqeunce needs to be removed)
Pre- mRNA = Have a 5’ splice site (5’ relative to teh intron) and a 3’ splice site AND have a branching point in the middle
- AGGU concecus sequence at the 5’ end (GU are the most important nucletdoes)
- CAAG concecus sequence at the 3’ end (AG are the improtant nucleptides)
- A n the middle of a poly pyridine track = branching point on the middle of intron
Cleavage occurs between the GG on the 5’ end and the GG on the 3’ end
Splicing Reaction
Overall - transferication reactions
- Splicing = occurs co-transcriptionally
Steps of reaction (rearranging 2 phoshodietser bonds):
1. The branching point A is attached b a 5’ Phosphate and a 3’OH BUT it has a free 2’OH that can do a nucleophilic attack –> 2’OH will attack the phophate bond between the end of the exon and the begining of the intron = forms a 2’-5’
- Makes a layreyet on the intron where intron is attached to itself
2. Upstream exon has a free 3’OH that will do a nucleophilic attack on the phosphodietser bond between the intron and exon 2 –> makes a new phosphate bond between the exon
- Causes relase of the 3’OH of introns
END - have ligated exons and a branched leriet 2’5’ intron
Gel of Splicing Reaction
Shows how people found order of events
ON gel - Have precursor at time 0 with 2 exons and introns –> then see free 5’ OH appear at the same time that exon 2 is joined to intron appears (first step of reaction) THEN see exon 2 appear at the same time as the lareit is released
Splisasome
Splisasome = ribonuclear protein machine
- Splisasome = protein directed ribozyme
Splisaome = maed up f 5 snRNPs + 100s of proteins
- U1,U2,U5,U6/U4 = control RNA polecules and proteins
- Splisome also uses 2 protein complexes (NTC and NTR)
Splcing Reaction
HAVE lots of rearrangement of RNA paring and breaking apart in order to bring ends together and catalyze reaction
Consequence of splicing
Consequence of splicing = EJC deposition
EJC = exon junction complex –> mark of correct splicing
- Found at the junction of the joining exons
- Splisome = brings proteins of EJC to a place where it catalyzes the reaction = leaves mark on mRNA that has been properly spliced
- EJC = deposited by the splisome itself
Use of EJC:
1. EJC = needed for export
2. Used for quality control processing required for translation
Splisasome + RNA world
Splisasome = reminenet of the RNA world
Splicing = catalyzed by the Splisasome BUT splciing orginated before the Splisasome existed
- Before splisasome = had self-splicing introns (Ex. group 1 and group 2 self splicing introns)
- Self splicing introns = 1st ribozymes found
Example - Group 2 = uses the same mechasnims as us BUT doesn’t require energy
Extreme case of alternative processing
Example 1 - Protein Dscam - detrmines nueronal adhesion
- Have alternative splicing at different cassettes (4 parts of the protein are ended by alternative exons
- Any 1 molecules is made of 1 green, 1 red, 1 blue, 1 purple (have 12 gteen exons + 38 red exons etc.) = have 38,000 splice isoforms that a cell can make
- Ensures all nuerons make a diferemt Dscam
Example 2 of splicing – Procadherins in humans – diversity is generated by using alternative initiation and alternative splicing
- Have 3 exos (ex. 15 purple) –> have different initiation sites (each has its own promoter) = get alternaive intiation
Use of Alternative splicing in Drugs
Did experiment - Run SMN1 and SMN2 in RT-qPCR = SMN1 makes full length product and SMN2 makes trunctaed product
THEN Run SMN2 with a mutation in the splicing silencer = get full form of SMN2 –> gave people idea to help SMA patients by blocking the silencer
NOW = give antisense oligos that are complementary to sequence in intron 7 -> ca turn SMN2 gene to make a full length product
Average half life of different RNA molecules
Half life of RNA is different in different types of RNA
- tRNA and rRNA can be stable for days vs. mRNA can be stable for minutes-hours
- Rate of decay is under strong conrtol
Bacterial mRNA decay
Bacterial mRNA decay = needs endonucleaic cleavage (Decay starts with enodnuclease cleavage)
BActeia ahve 3’ end and 5’ triphophate structure
Eukaryotic mRNA decay
Overall - Begins with deadenylation then decapping then exonucleaic cleave from the ends by XRN1 and exosome
- miRNA and other proetins intiate process
Process:
1. To degrade Eukaryotic mRNA = polyA tail needs to be degarded = need to remove the polyA protein and the deadenylate
2. Once have a short polyA tail = get decapping
- Decapping = cleaves the 5’5’ phosphate bond) = allows exonuclease to chew mRNA from ends
Hypothesis for evolution of complexity
Hypothesis for evolution of complexity = arms race between nucleic acids trying to invade genomes and the body coming up with solutions to combat this
- Arms race = led to additions to gene regulatory process –> generates diverse patterns of gene expression –> can evolve more complexity
- Likely evolved as part of defense against genomic parasites
Example – nuclear envelope and chromatin = protect DNA from transposons and viruses –> THEN once tranpsosons and viruses could get in needed to change again
- Once have nuclear envelope = need t get mRNA out
