RNA processing in Eukaryotic Cells

When does RNA processing occur (in relativity to transcription and translation)? Where does it occur

happens after transcription but before translation in eukaryotes

in the nucleus

What 3 things occur during RNA processing in eukaryotic cells to mature mRNA?

1) 5'mRNA capping (7meGppp)

2) poly A tail addition and cleavage (3' end processing)

3) Splicing of introns

What is the goal of RNA processing (aka what does the final transcript or mRNA look like)

has 7meGppp 5' cap, polyA tail and no introns (still has 5'UTR, ORF, and 3' UTR like in prokaryotes)

What is the general mechanism for 5' mRNA capping?

1. Removal of terminal Pi (recall that there are 3 P) from 5' end of nascent (newly synthesized) RNA transcript

2. Guanine cap is attached to 5' end using GTP (GTP loses 2 phosphates)-> this is a 5'to5' linkage!!

3. Methylation of guanine and some ribose

the cap is methylated guanosine (has only one phosphate)

What is the overview of Assembly on CTD (more specifics on 5' mRNA capping)

1. Transcription factor TFIIH kinase phosphorylates CTD ( c terminal domain) of RNA polymerase II on Ser5 of repeat

2. Stall factor stops PolII shortly downstream of initiation site so capping happens before RNA poly continues

3. Capping enzymes bind to Ser5-phosphorylated CTD and adds cap to 5' end of mRNA

4. A second kinase phosphorylates stall factor and Ser 2 of repeat on CTD

5. phosphorylated stall factor is removed and transcription can continue because RNA poly is released

6. Additional factors are recruited to Ser2-phosphorylated CT that facilitate splicing and passage through downstream chromatin

What are the functions/purposes of 5' mRNA capping?

1. Increase transcript stability by protecting against ribonuclease (which chew up DNA via phosphodiester linkages 5'->3' but the cap has a triphosphate linkage that ribonuclease can't chew up)

2. Increase translation efficiency because 5' cap binds to translation initiation factors

How is the polyA tail added to mRNA?

1. Cleavage signal on nascent RNA is recognized by endonuclease (context or position of signal matters, otherwise cleavage won't happen)

2. Endonucleases cleave about 20 bases off end

3. Add A tail by polyA polymerase (get A's from ATP but you lose 2 phosphates) -> untemplated

What is the function of the polyA tail? What happens if you don't have polyA synthesis?

It may be involved with stability of mRNA so ribonucleases can't cleave other end and enhances translation efficiency

You still get primary transcript synthesis and mRNA without tail can still be transported out of nucleus, but protein synthesis is less efficient

What are introns and exons? What is the difference in size between the two? Does mutation in intron region lead to an effect on protein?

Introns are RNA sequences that aren't present in processed/mature mRNA

Exons are sequences that are present in processed mRNA

Introns are large compared to exons and higher/more complex organisms are more likely to have introns, and have more of them and larger introns (in humans, most of gene is introns)

no!

What is the overview of intron splicing?

mRNA precursor recognizes intro and exon and marks 5' and 3' splice sites (5' splice site and 3' splice site references intron) and then splicing occurs so there is just exons spliced together and excised intron

What signals are needed at the splice site of introns?

On 5' splice site: ...(cut)GU... (right after cut)

On 3' splice site: ...AG(cut).... (right before cut)

On branch point: A (in the middle)

What are the two steps in splicing out introns? What is the result

(both called trans-esterification reactions where phosphodiester bonds are broken and remade)

1. First transesterification reaction: Involves branch point and 5' splice site

-2' OH group of A on branch point will attack phosphate at 5' splice site which results in a 5' hydroxyl on exon

2. Second transesterification reaction: involves 3' splice site and 5' splice site (what remains)

- the 5' OH attacks phosphate at 3' splice site

the result: a lariat (the intron) and the exons spliced together

What is the spliceosome? What ingredients do you need to build it?

Does cutting/ splicing

-small nuclear RiobNucleo Proteins (snRNPs) which are RNA/protein complexes: U1, U2, U4, U5, and U6

What are the steps to assemble a spliceosome?

1. Splicing begins with recognition of 5' splice site by U1 snRNA in U1 snRNP

2. A protein complex is formed at 3' splice site which recruits U2 snRNP to branch point

3. U4, U5, and U6 complex binds

4. U1 and U4 leave spliceosome which leaves the U2, U5, and U6 which actually do transesterification steps

What directs and catalyzes mRNA splicing?What exactly does each snRNP do in splicing?

- snRNA in snRNPs

1. U1 binds to 5' splice site and U2 binds to branch point, U5 binds to bring them together

2. U4 and U6 bind to spliceosome, and when U4 leaves, U6 is released

3. U6 base pairs to U2

4. U6 displaces U1 at 5' splice site

5. Exons are then clipped and ligated and lariat (intron is released)

Why does alternative splicing happen? What pattern of alternative splicing do we have to know?

B/c not 100% consensus of splice sites and branch point; Exon inclusion/exclusion (sometimes exon is cut out too with removal of introns)

Why must exons be defined? How do you separate intron from exon?

So that it does not appear to be part of intron

exons defined by interactions between proteins bound to 3' and 5' splice sites and exon-bound proteins (first and last exons will have 5' cap bound proteins and polyA signal bound proteins)

U1 binds to 5' splice site and U2 and protein complex binding to branch point and 3' splice site respectively are important part of distinguishing exons from introns

We said that if splice site is not consensus, then the there is weaker binding of the snRNPs- more likely to result in alternative splicing pattern. How can this be fixed?

Exon Splicing Enhancer elements (ESE's) which bind serine/arginine rich (SR) proteins through RNA recognition motifs which provides additional contacts to recruit protein complex and/or U1

What is the structure of SR protein?

It is a tether and an activation domain to aid in recruitment of splicing machinery (one part of tether has RNA recognition motif and the other part of there is domain where splicing machinery can bind)

How do you account for regulated or cell-type specific exon inclusion or exclusion pattern of alternate splicing?

Through regulation( activator dependent inclusion, repressor dependent exclusion)

What is activator dependent inclusion? What happens if activator is present? Absent?

Requires a weak 5' or 3' splice site which leads to requirement of ESE and SR proteins

if activator is present, exon is defined and included in mRNA

if activator absent, then u1 won't bind which means exon not defined

What is required for repressor dependent exclusion? What happens if repressor is present? Absent?

5' and 3' splice sites are good (have consensus sequence) but presence of repressor determines if it is defined

if repressor present, U1, U2 and PC won't bind so exon not defined and is excluded

if repressor absent, exon is defined and exon is included

How are transcription and splicing integrated?

Components of splicing machinery are carried by phosphorylated poly II CTD

Splicing is cotranscriptional

splicing dependent on chromatin structure and polymerization rates

What are self splicing introns? What are the two types?

intron RNA folds into a structure that catalyzes its own removal (RNA functions as enzyme or ribozyme)

Group 1

Group 2

What are group 1 self splicing introns? Group 2?

Group 1: Intron with 3'-OH guanine nucleoside attacks 5' phosphate at 5'SS of mRNA (has 5'/3' ligated exons

Group 2: mechanism is same as spliceosome catalyzed mechanism in which proteins are involved (but not snRNPs)

How are eukaryotic ribosomes different from those of prokaryotes?

Eukaryotic ribosomes have larger rRNA (extra component) and have more proteins

subunits are larger: 40s+60s= 80s

How is translation initiated in eukaryotes?

1. The 5' mRNA cap binds initiation factors (eIF's) and recruits additional eIFs (with GTP), 40 s subunit and met-tRNA

2. 40 s subunit (with initiator methionine) scans mRNA to first AUG (movement requires ATP)

3. met-tRNA is positioned over the AUG, 60s subunit binds to 40s subunit, GTP is hydrolyzed and eIFs are released

How is regulation different of translation different in prokaryotes than eukaryotes?

Prokaryotes are regulated by access to Shine-Delgarno Sequence and one gene at a time

Eukaryotes are regulated many genes at a time through translation factor that target the 5' cap and is regulated by microRNAs (small RNAs) that bind sequences in 3' UTR of target genes and inhibit translation

What are microRNAs and what do they do?

They are small RNAs that bind sequences in 3' UTRs of target genes to inhibit translation

How are microRNAs (aka miRNA) formed?

1. Transcribed by Polymerase 2 as a 5' capped and 3' polyA noncoding RNA which forms a hairpin structure (pri-microRNA)

2. pri-microRNA is processed by Drosha (a ribonuclease) which releases hairpin structure (result is pre-microRNA)

3. premicroRNA exits nucleus and goes to cytoplasm

4. Dicer (a ribonuclease) cuts off loop which releases microRNA in cytoplasm

5. RNA induced Silencing complex (RISC) chooses one of strands and presents it to mRNA for complementary base pairing and translation repression

What does imperfect base pairing of miRNAs with target sequences with 3' UTRs of mRNA result in?

Translation repression

What happens to proteins after translation?

1. Folding into correct 3D structure and assembly into complexes

2. Targeting for secretion or to different compartments of the cell

3. Covalent post-translation modifications

How are proteins targeted?

Through their addresses, which are short amino acid sequences that target them to different locations and are called signal sequences

What are 3 ways proteins are moved within a cell?

1. Through gated transport of folded protein through hole or pore

2. Transport of unfolded protein across membrane

3. Transport through vesicles between different compartments?

How is protein transported to the nucleus?

Through way 1 (gated transport of folded protein through hole or pore)

Large molecules like these proteins cannot just freely diffuse through pore, so they have nuclear localization signal (NLS) that binds to nuclear import receptors which will enable transport through the Nuclear Pore Complex (unidirectional movement and need GTP)

How do you transport proteins to ER ?

1. Signal peptide emerges from ribosome and is bound to signal recognition particle (SRP) so translation stops momentarily

2. SRP binds to SRP receptor so translation can continue with polypeptide going into ER Lumen

3. SRP is cleaved from polypeptide and translation continues

4. After translation complete, ribosomes dissociate

proteins destined for secretion synthesized into ER where they will undergo vesicular transport to leave cell (translation and transport are coupled to get protein across ER membrane)

What are the ways in which protein can be modified?

1. Phosphorylation: add phosphate group

2. Methylation: add methyl group