poly(A) tail

1. how many and to what end

2. what protein sits on tail

3. two functions

4. what adds the adenosines

1. 100-250 adenosines to 3' end of mRNA

2. associated with poly(A) binding protein

3. important for nuclear export, translation regulation, and stability of mRNA

4. poly (A) polymerase

Splicing consensus sequences

1. GU at start of intron

2. branch A 20-50 bp from 3' end

3. 15 bp pyrim. rich region btw A branch and 3' end

4. AG at end of intron

splicing process

spliceosome mediated (5 snRNPs bind, loop pre-mRNA)

two transesterfication reactions

result in attached exons

lariat intron excised

EJC is added to mRNA

amino acid structure

linking amino acids to form polypeptides

two methods of transcriptional termination in prokaryotes

rho dependent

rho independent

rho dependent transcriptional termination in prokaryotes

rho-independent transcriptional termination in prokaryotes

there are elements in the template DNA that result in hairpin loop in new RNA strand followed by a run of Us

this destabilizes RNAP and makes it detach from the DNA template

method of transcription termination for Pol I

method of transcription termination for Pol III

like Rho-independent transcription termination in prokaryotes

RNA hairpin structure destabilizes complex--smaller run of Us

transcription termination in Pol II

two models of how the Poly(A) site triggers termination

1. torpedo model

2. allosteric model

torpedo model

cotranscriptional cleavage of nascent transcript at poly(A) signal

provides entry site for 5'-3' exonuclease called Rat1 in yeast and Xrn2 in animals

Rat1 in yeast and Xrn2 in animals

5'->3' exonuclease that chews up rest of nascent RNA chain after cleavage at poly(A) site

helps terminate transcription (torpedo model)

disruption of this protein results in RNAPII transcribing beyond regions where it normally terminates

allosteric model

of RNA Pol II

transcription termination

poly (A) site causes change in elongation complex which increases its tendancy to terminate

(change in the EC could involve release of a factor that normally inhibits termination or recruitment of a factor that causes termination)

what is the 5' cap addition on newly synthesized mRNA?

7-methylguanosine

(m7G)

has three phosphates

how is 5' cap attached on new mRNA strands?

the 7-methylguanosine is inverted

there is a 5' to 5' triphosphate bond

the addition of the cap occurs after ~20 to 30 nucleotides have been synthesized by RNAPII

does RNA transcribed from Pol I and Pol III have 5' cap?

three important functions of 5' cap

1. transport

interacts with cap-binding complex (CBC)

2. protection

prevents 5'->3' exonuclease digestion

3. activity (ribosome docking)

Two poly(A) signals

1. AAUAAA which is 10-35 nucleotides upstream from poly(A) tail

2. GU rich or U rich region downstream of cleavage site

CPSF

cleavage and poly(A) specificity factor

binds AAUAAA site

on newly synthesized mRNA; involved in polyadenylation

CStF

cleavage stimulatory factor

involved in polyadenylation on new mRNA

binds G/U site and interacts with bound CPSF forming loop

PAP

Poly(A) polymerase

binds to new mRNA strand and links cleavage and polyadenylation

PABPII

responsible for what three things

and what does it stand for

poly(A) binding protein

(localized in nucleus)

1. second phase (fast phase) of polyadenylation by PAP

2. facilitating mRNA export into cytoplasm

3. termination of polyadenylation by PAP

on average, a eukaryotic gene contains around how many introns per kb of protein coding region?

~4

purpose of introns?

alternative splicing--allows for protein diversity

exon shuffling--allows for evolution of new genes

ribonucleoprotein

nucleoprotein that contains RNA

snRNPs

small nuclear ribonucleoproteins

spliceosome is assembled from proteins and RNAs called snRNPs

splicesome assembly requires what?

ATP hydrolysis

spliceosome components

pre-mRNA

5 snRNPs (U1, U2, U4, U5, & U6)

and over 100 protein factors

EJC

exon junction complex added just upstream of where two exons have been joined after an intron has been removed

how cells keep track of where they spliced

serve as regulator in Nonsense mediated decay

intron length

typical length

minimum length--what dictates this?

typical length: 40-150 nt long

minimal length: ~20 nt

dictated by ability of splicing machinery to assemble on the intron

typical exon size distribution

50-300 nt

alternative splicing provides means of

regulating gene expression

increasing protein diversity

trans-splicing

method used in C. elgans and other nematodes to give different exons that were spliced out of an operon 5' caps so they can make their own proteins

SL

stands for

where does it go

what does it come from

spliced leader

sequence trans-spliced onto 5' end of exons from an operon in nematodes

SL sequence donated by a 100 nt snRNP

RNA editing

two distinct mechanisms

1. Substitution editing

2. insertion / deletion editing

substitution editing

catalyzed by enzymes that recognize a target sequence (like restriction enzymes)

cytidine deaminase (C->U)

adenosine deaminase (A->Inosine, which ribosome translates ->G)

Insertion/deletion RNA editing mechanism

alterations mediated by guide RNA molecules that base pair with RNA to be edited and serve as a template for addition or removal of nucleotides in the target

apoB gene

1. what does it do

2. example of what

3. where does the special processing occur (tissue and exon number)

4. what happens there

5. different results in two tissues

1. encodes two alternate forms of a serum protein central to uptake and transport of cholesterol

2. example of RNA editing-by substitution editing

3. which occurs in the intestine--exon 26 out of 29

4. cytidine deaminase changes CAA->UAA (stop codon)

5. in intestine, ApoB-48 ~2k a.a.

in liver, ApoB-100 ~4.5k a.a.

how many average nuclear pore complexes per cell?

average 2000

proteins that make up the nuclear pore complex are known as

nucleoporins

hnRNPs

heterogeneous ribonucleoprotein particles

there is a nuclear protein component

and a nuclear RNA component

contribute to splicing, polyadenylation, export

bind to nascent mRNA strand to form complex mRNP

mRNPs

messenger ribonucleoprotein particles

fully processed mRNAs in nucleus remain bound by hnRNPs in complexes referred to as mRNPs

the assembly of this complex on nascent mRNA generates a stable and export-competent mRNP particle.

what cytoplasmic mRNA conformation is key for translation competency

and what cytoplasmic proteins and mRNA components make this conformation possible?

allows for cluster of what--give name

circular conformation

PolyA tail has to be long enough for a few PABPs to bind

PABPs help stabilize elF4E, which bind 5' cap

(via it's interations with elF4G)

alows for cluster of ribosomes attached to same mRNA--polysome

Is translation possible without mRNA 5' cap?

1. what is the mechanism

2. often used by what

3.what does it do to normal translation machinery

4. used in healthy cell for what

1. IRES

2. often used by viruses

3. starts translation of viral mRNA and shuts down translation in host cell by cleaving eiF4G so that it cannot interact with elF4E

4. cell uses IRES during mitosis and programmed cell death

IRES

internal ribosome entry site

nucleotide sequence that allows for translation initiation in middle of mRNA

What induces conformational change that helps in translocation of ribosome along mRNA and in peptide bond formation?

hydrolysis of GTP

cytoplasmic poly(A) regulation

give example

give protein interactions on translationally dormant mRNA and translationally active mRNA

1. in oocytes--

some of the stored mRNAs have short poly(A) tail--don't get translated until egg is fertilized

in response to external signal A residues are added, stimulating translation

2. dormant: CPEB / Maskin / elF4E

active: PABPI / elF4G / elF4E

PABI

polyadenine binding protein I

located in cytoplasm

helps form circular conformation of mRNA during translation

multiple PABIs bind to poly(A) tail and interact with elF4G--stabilizing interaction of 5'cap with elF4E which is required for translation

CPEB

cytoplasmic polyadenylation element binding protein

CPEB most commonly activates the target RNA for translation, but can also act as a repressor,dependent on its phosphorylation state.

on dormant mRNA it interacts with maskin which interacts with elF4E, represses translation

nonsense mediated decay

quality control mechanism that

1. detects premature stop codons on mRNA

2. triggers them for decay so bad proteins aren't made

searches for EJC downstream of stop codon

if yes, triggers decay

three mechanisms of RNA degradation in the cytoplasm

1.decapping pathway (5'->3' exonuclease)

2.deadenylation pathway (can result in exonucleatic decay from both directions)

3. endonucleolytic pathway (results in 3'->5' exonucleolutic decay)

mechanism of siRNA targeted degradation

endonucleolytic pathway

cleave in middle followed by 3'->5' exonucleolytic decay

deadenylase

enyzme that catalizes poly(A) shortening and deadenylation dependent RNA degradation pathway

exosome

protein complex involved in RNA degradation

degrades in 3' -> 5' direction

Several proteins that stabilize or destabilize mRNA molecules through binding to AU-rich elements in the 3' untranslated region of mRNAs interact with the exosome complex

RISC

RNA-induced silencing complex

RITS

nuclear form of RISC

RNA-induced transcriptional silencing complex

Ferritin

high iron

low iron

controls storage of iron in cells

IREs in 5' UTR of ferritin gene bound by IRE-BP as form of translational control)

high iron--IREs unbound--ferritin translated (prevents harmful accumulation)

low iron--IREs bound--no transl initiation

(decrease in ferritin in cell)

IRE

iron response element

sequences where RNA binding proteins (IRE-BP) bind

5' end of ferritin mRNA to control translation

3' end of transferrin mRNA to control degradation

IRE-BP

when bound to 5' UTR

3' UTR

bound or unbound during high iron levels?

regulates level of free iron ions within cells

when bound to 5' UTR of ferritin mRNA--REPRESSES translation

when bound to 3' UTR of transferrin mRNA--PROTECTS against degradation

in both examples, in cases of high iron IRE-BPs DON"T bind

Transferrin

high iron

low iron

controls import of iron into cells

mRNA of transferrin receptor has IREs whose stems are rich in A-U regions, destabilizing sequences

high iron: mRNA degraded

low iron: mRNA protected against degradation

cytoplasmic mechanism of post-transcriptional control

RNAi

RISC

1. stands for

2. found in which pathways

3. what does it do

4. known components

5. can one RISC bind to repress translation?

1. RNA-induced silencing complex

2. both siRNA and miRNA assembled into RISC complexes

3. mediates recognition and hybridization btw small RNA and target mRNA--cleaves mRNA

4. known components: Dicer, Argonaute prots, various RNA binding prots

5. inhibition of target mRNA translation requires binding of 2 or more RISC complexes

Argonaute proteins

two domains and domain functions

proteins that are components of RISC complex

in RNAi pathway

AGO1 and AGO2 contain

PAZ domain (binds small RNA)

C terminal PIWI domain (recognizes dsRNA)

miRNAs

binds where on mRNA

what does it do to target mRNA

bind 3' UTR of mRNA

triggers accelerated deadenylation and decapping of partially complementary targets

origins of miRNA

1. precursors

2. what is length of mature miRNA

3. proteins that act on it or with it in nucleus, for transport, and in cytoplasm

1 A. originate from capped & polyadenylated full length precursors (pri-miRNA)

1 B. hairpin precursor ~70 nt (pre-miRNA)

2. final length ~22 nt

3. Drosha and Pasha create hairpin pre-miRNA

Exportin 5 helps export it

Dicer cuts duplex down to appropriate size

RISC assembly--gets bound by Argonaute

Examples of how miRNA mode of post-transcriptional regulation is useful

early development (no transcription--need method for regulation)

motor neurons (very long, sometimes meters; make RNA send it all down--repress where you don't need it)

reinforcers / back up plan fail safe mechanism to ensure accuracy; back up plan if problem with transcription regulation machinery

lin-4

lin-4 supresses LIN-14 protein which prevents L2-type cell divisions

mechanism of action: lin-4 inhibits translation of lin-14

example of miRNA regulation in C. elegans

let-7

C. elegans miRNA

example of miRNA regulation in C. elegans

inhibits lin-14 and lin-41, which prevents lin-29 translation and generation of adult cell lineages

lin-29

mRNA in C. elegans which helps with generation of adult cell lineages

inhibited by lin-41

which small RNAs are genetically encoded?

miRNAs

siRNAs are produced how

exogenously

or

from bidirectionally transcribed RNAs

main difference btw miRNAs and siRNAs

complementarity

result of mRNA targeting

miRNAs imperfectly bind

inhibit translation

siRNAs perfectly bind

trigger degradation

fate of mRNA loaded with the miRISC

mRNA stored in P bodies

where it undergoes degradation or it is sequestered from translation and could possibly reenter translation pathway

p bodies

processing bodies

area in cytoplasm that contains many enzymes involved in mRNA turnover

place known to degrade and decap, store, translationally repress mRNAs

involved in miRNA pathway

miRNA target prediction

sequence before structure methods

many initial algorithms share common approach

1. evaluate miRNA target complementarity first

2. then move to evaluating binding site thermodynamics

3. target pool filtered by requiring species conservation

miRNA seed match

complementarity btw 6 or 7 5' nucleotides of an miRNA and the 3' UTR of its target mRNA

bind perfectly

RNAi and inhibition of transcription

1. name of pathway

2. steps

1. repeat associated siRNA (rasiRNA) pathway

2. a. transcription of certain seqs leads to long dsRNAs

b. cleaved by dicer into siRNAs

c. single strand taken up by RITS

d. RITS directs DNA and histone methylation and recruits other heterochomatin-associated proteins ro region of genes with same sequence as siRNAs

rasiRNA

repeat-associated siRNA pathway

inhibition of transcription via heterochromatin formation using RNAi pathway

transcription from what type of DNA leads to siRNAs in rasiRNA pathway?

transcription from opposing promoters found in repetitive DNA elements (such as centromeric regions and satellite DNA)

RITS

what does it stand for

what is it required for

RNA-induced transcriptional silencing complex

required for spreading of H3K9 methylation in peri-centromeric repeat regions

CBC

cap-binding complex

helps mRNAs get exported from nucleus

(illustrates importance of 5' cap)

name five modes of alternative splicing

1. skipping/inclusion

2. mutual exclusion

3. alternative 3'

4. alternative 5'

5. intron retention