EPIGENETICS EXAM 2

Overview of RNA mediated silencing

Summary of RNA Biogenesis

No RdRP in vertebrates and insects

Core features of siRNa and miRNA silencing

What are two main pathways of RNA mediate silencing?

1) RISC
2) RITS

RISC

RNA induced silencing complex

RITS

RNa induced transcriptional silencing

Where does RISC occur?

cytoplasm

RISC

Argonaute mediated is _________________silencing 

post transcriptional

RISC

_________ and ________recognize mRNA target

miRNA and siRNA

RISC
Degradation of the paired region via ____________________ (hydrolyzing bonds between nucleotides)

endonucleolytic cleavage

RISC

The________________ domain of the argonaute/PIWI family protein (a subunit of RISC) performs this initial mRNA cleavage event.

RNase H

RITS

_________stops the transcript from being synthesized

transcriptional

RITS

works through __________alterations

chromatin

RITS

_________and other protein components target the complex to chromosome regions fro chromatin modification

siRNAs

____________recognizes a perfectly complementary mRNA.

siRISC

______________________mRNA cleavage at a single site within the duplex

Ago-catalyzed

Slicing is very precise-___________________between the target nucleotides that are based paired to siRNA residues 10 and 11 (counting from the 5’ end) is cleaved generate products with 5’ monophosphate and 3’hydroxyl termini.

phosphodiester linkage

______________________attack the cut fragments to complete the degradative process.

cellular exonucleases

After cleavage, functional siRISC is______________.

regenerated 

siRNAs are capable of recognizing targets with imperfect _____________ (upper right).

complementarity

siRNA can silence targets by miRNA like mechanisms involving ________________ and ______________________.

translational repression and exonucleolytic degradation

Non Expressed mRNAs recruit _______________ and ______________________and form circularized structures that enhance translation.

initiation factors and ribosomal subunits

ribosomal recruitment

elF4f

biogenesis and maturation of 60S ribosomal subunits

elF6

miRISCS bind to mRNAs and repress initiation at the _____________ stage or the ___ recruitment stage

cap recognition; 60s

miRNA can induce _____________of mRNA and thereby inhibit circularization of the mRNA

deadenylation

miRNA can also repress a __________________stage of translation by inducing ribosomes to drop off prematurely 

post initiation

miRNA can promote ___________________ by inducing deadenylation followed by decapping.

mRNA degradation

_______________ heterochromatic regions have specific structure.

Constitutive

Pericentromeric regions are transcribed ______________.

bidirectionally

These repeats allow formation and maintenance of heterochromatin by proving source for ____________________.

double stranded RNA

co transcriptional gene silencing in Yeast S. Pombe

• RdRC=RNA dependent RNA polymerase

• Rdp1=RNA dependent RNA polymerase 1

• Dcr=Dicer 1

• Ago1=Argonaute 1

• Chp1=Chromodomain Protein that binds to H3K9

• CLRC=Cryptic Loci Regulator Complex-causes H3K9 methylation via Clr4

• Swi6=heterochromatin protein 1 (HP1) ortholog

• Clr4=H3K9 methyltransferases

Pericentromeric repeats produce long ___________by RNA directed RNA polymerase complex (RCDC)

ncRNA

siRNA are produced from ______ targeted transcripts

Ago1

These are processed into _________ by dicer

siRNAs

Double stranded siRNA are loaded onto __________________________and converted to single stranded siRNA. 

Argonaute chaperone complex (ARC)

__________________ initiates transcription at loci targeted by RNA interference (RNAi)

RNA polymerase II

During the ______________ phase of transcription, the Argonaute Ago1 is guided to nascent transcript and inhibits RNA poly II transcription by an unknown mechanism.

elongation

RNAi can lead to the release of __________.

RNA Pol II

RITS complex includes _______and ________: chromodomain protein that can guide the complex of H3K9me

ARC and Chp1

RITS complex can now target newly formed transcripts based on __________________.

siRNA homology

RITS complex recruits H3K9 methyltransferase ___and causes H3K9me.

Clr4

___________then attracts Swi6 (Containing chromodomain)

H3K9me

____________recruits RDRC and promotes double stranded RNA synthesis. This leads to fully silenced heterochromatin.

Swi6

Co transcriptional silencing by siRNA in plants

______acts early in RdDM pathway-produces 24 nt siRNA. Targets are mostly transposons and other repeats.

Pol IV

Pol IV-interacting protein SHH! likely recruit IV to it’s targets. SHH1 binds to _______ and methylated ________.

H3K9me and H3K4

CLSY is a__________ATP dependent remodeler

SWI/SNF2

siRNAs are methylated by _____to stabilize the siRNA

HEN1

SiRNAs are then loaded to Argonaute _________.

AGO4

_____is involved in RdDM. It helps locate the siRNA-AGo4 complex to target loci.

Poly V

_______transcripts attract the complex the complex by base pairing with siRNA

Pol V

______ complex is required for pol V transcription and for RdDM. 

DDR

What are 3 components of DDR?

• DRD1

• DMS3

• RDM1

_______is a SWI2/SNF2 chromatin remodeler

DRD1

_______is a single stranded DNA binding protein with a preference for binding methylated DNA

RDM1

__________is similar to proteins involved in structural maintenance of chromosomes. 

DMS3

_______ likely recruits DRM2 and cause de novo DNA methylation

RDM1

DRM2 causes de novo ______,_______,_____ methylation

• CG

• CHG

• CHH

_____is the only known pathway to CHH methylation

RdDM

What is the nucleic acid structure? Functions?

deoxyadenosine 5’-monophosphate (dAMP)

functions:

• store and transmit genetic information

• DNA and RNA are made from nucleotide building blocks

• Nucleotides comprise a base, sugar, and phosphate

• nucleotides have additional biological functions, such as a energy storage (ATP) and molecular transport

Nucleic Acid Structure: DNA vs RNA

DNA and RNA two types of sugars found in nucleotide with both purine and pyrimidine bases. What is the main difference between the two? 

DNA: 

• contains deoxyribose

• Adenine, guanine, cytosine and thymine

• double stranded

RNA:

• contains ribose and has an additional oxygen atom on carbon 2. 

• no thymine, but uses uracil instead

• single stranded

• can base pair with DNA U w/A

Each base is joined to a sugar by a _____________ between _____ of sugar and the _______ of a pyrimidine or______of a purine.

glycosidic; C1; N1; N9

Base plus sugar is called what?

Nucleoside

• Adenosine

• Guanosine

• Cytidine

• Thymidine

• Uridine

Nucleoside plus phosphate is a nucleotide. Phosphate groups are linked to the ____of sugar.

C5

____________are RNA’s that do not directly code for polypeptides

NonCoding (nc) RNA

__________ and ________________are among the shortest and involved in the RNA induced silencing complex 

MicroRNAs and small interfering RNAs 

_____________facilitate chemical modifications to rRNA in the nucleolus

Small Nucleolar RNAs (snoRNAs)

_________________are larger than 200nt, that regulate mRNA expression both positively and negatively

Long non-coding RNAs (incRNAs)

________________control many biological processes (not directly carrying out a process) in all kingdoms of life. This is in contrast to tRNA, rRNAs, snRNA’s that are directly involved in the process.

Regulatory RNAs

The diverse roles of regulatory RNA's are only just becoming apparent. 

______of the human genome is transcribed, but only ____ has ORFs. 

60%; 2%

What are the three main principles that appear to hold true for many regulatory RNA molecules? 

1. primary transcripts of regulatory RNA molecules are often processed to yield final functional molecule

2. Regulatory RNA molecules are often processed to yield the final functional molecule

3. Regulatory RNA’s often interact with other components (like proteins) to carry out their functions

Regulatory RNA’s usually interact with targets via_______________.

base pairing

What are the 3 effects of base pairing interactions?

1. disruption of protein binding

2. alteration of RNA structure

3. recruitment of proteins

How are regulatory RNA’s (green) encoded in different ways with respect to the target (blue)

a) on the DNA strand antisense to the target

b) in a completely separate DNA region (trans)

as part of the target (cis)

What does the flexibility of RNA structure allow for in regulatory RNA’s?

It allows for binding to molecules other than DNA/RNA like proteins and metabolites.

_______are a pool of 20-30 nt RNAs that generally function in gene silencing.

Small RNAs

Small RNAs contribute to _____________________ by affecting mRNA stability or translation.

Post Transcriptional Gene Silencing

• stops before transcription, heterochromatin and RNA polymerase can’t bind

Small RNA’s contribute to transcriptional gene silencing through __________________to chromatin. 

epigenetic modifications

RNA silencing uses a set of core reactions. What are they?

1) Dicer/dicer like proteins

2) Argonautes

Double stranded RNA dsRNA is processed by ________or DCL protein into short RNA duplexes.

Dicer

These small RNAs subsequently associate with ___________________proteins to confer silencing.

Argonaute (AGO)

Which RNAse family are dicer and dicer like proteins in? 

RNA III family

In which biogenesis do dicer or DCL proteins cleave long dsRNA or foldback (hairpin) RNA?

siRNA and miRNA

How long are the fragments that dicer or dcl porteins cleave in biogenesis.

21-25 nt

Dicers structure allows it to measure the RNA it is cleaving. Like a cook who dices a carrot, dicer chops RNA into_____________pieces.

uniformly sized

______________enzymes are involved in miRNA and siRNA processing.

RNAse III family

__________________domain cuts double strand in two places

dimeric catalytic

___________________enzymes must dimerize, but Drosha and Dicer already contain two catalytic domains and can act as a monomer.

Class I

_______________proteins bind to small RNAs and their targets.

Argonaute (AGO)

In which organism was Arabidopsis ago1 mutant discussed in in class? 

Octopus (argonauta argo)

AGO proteins are named after the argonaute1 mutant of arabidopsis; ago 1 has thin radial leaves and was named for octopus for which it resembles.

_______________derived from primary transcripts and generally downregulate cytoplasmic RNA’s through translation repression and mRNA decay (postranscriptional)

microRNA (miRNAs)

_________________are derived from longer double stranded RNAs (viral or endogenous RNAs).

small interfering RNAs (siRNAs)

Why is it important that siRNAs target RNA’s for degradation?

because it helps them to acts as cellular defense mechanism

___________________production is triggered by miRNA and AGO mediate cleavage of dsRNA transcripts originating from TAS loci. 

trans-acting siRNAs (tasiRNA's)

What is the role of tasiRNAs?

They help regulate non identical mRNAs as well as miRNAs.

_____________________are deprived from repetitive regions of the genome and downregulate transcranscriptio from repetitive regions

repeated associate small interfering RNAs (rasiRNAs)

What are examples of repetitive regions in rasiRNAs?

hcRNAs, piRNAs

All eukaryotic _______ associate with Argonaute proteins.

sRNAs

MicroRNAs (miRNAs) are derived from ___________________.

endogenous primary transcripts

Small interfering RNAs (siRNAs) are derived from ______________________________,

double stranded RNAs (viral or endogenous RNAs)