Week 11 - micro RNA and RNA-epigenetic

Question 1

Heterochronic genes

Answer 1

mutants affect timing of divisions

Question 2

lin-14 (lf)

Answer 2

lineage is disrupted; precocious development of L2 lineage
- 2 rounds of division in L1 larval stage

Question 3

lin-14 (gf)

Answer 3

suppression of L2 lineage and replacement with L1 lineage
- continuous reiteration of L1 lineage

Question 4

lin-4 (lf)

Answer 4

• result in same suppression of L2 lineage as Lin-14 gf
• continuous reiteration of L1 lineage

Question 5

Double mutants;
lin-4 (lf) lin-14 (lf)

Answer 5

demonstrated lin-14 phenotype

Question 6

Lin-4 ____ lin-14

Answer 6

represses

Question 7

lin-14 encodes a _______

Answer 7

nuclear protein

Question 8

lin-4 encodes a _______

Answer 8

non-coding RNA, a miRNA the first found

Question 9

lin-4 RNA ____ with Lin-14 _______

Answer 9

base pairs, 3’ UTR

Question 10

in lin-14 gf mutants, ________ are deleted

Answer 10

lin-4 binding sites

Question 11

lin-14 mRNA is expressed ______ larval stages, however lin-14 protein is expressed in _____ larval stages

Answer 11

throughout, L1

Question 12

lin-4 mRNA is expressed at ______ larval stages

Answer 12

L2, L3 and L4

Question 13

Let-7 and Lin-29

Answer 13

required for suppressing L4 events
- let7 (lf) and lin-29 (lf) depicts lineages dividing past adult stages
- lin-29 stops juvenile lineages from occurring and establishes adult lineages

Question 14

let-7 encodes ____ and can bp at the _____ of RNA

Answer 14

non-coding RNA, 3’ UTR

Question 15

miRNAs

Answer 15

• everywhere
• very short; 20 odd bases long
• sequencing 20 base RNAs uncovered many miRNAs

Question 16

miRNA transcription

Answer 16

• transcribed by RNAPII
• processed to form pri-miRNA
  
  • PASH-1 and DRSH-1 bind
• formation of hairpin structure = pre-miRNA –> EXPORTED TO CYTO and processed by dicer
  
  • forms 20 nucleotide miRNA that will form a complex with AGO where complex searches for mRNA that contain complementary RNA sequences
• RNA-induced silencing complex (RISC) forms –> brings silencing of translation of message
• miRNA turnover = miRNA have a lifespan
  
  • will be POLYURINATED –> uracils added to 3’ end for degradation

Question 16

miRNA suppresses ________

Answer 16

translation

Question 17

RNAi degrade _____ through _____

Answer 17

mRNA, cleavage

Question 17

miRNA guide ____ to ____ and ___ suppresses _____

Answer 17

RISC,
specific mRNAs,
RISC,
translation

(MODEL SUGGESSTS miRNA HAVE NO ROLE IN ACTIVATING RISC)

Question 17

Experiment to understand miRNA role in RISC proteins

Answer 17

1) tether AGO to RNA binding protein (ex: lambda N protein -> recognized b -box)

2) Two experiments,
1. mRNA with no b-box = TRANSLATION

  2. mRNA with b-box that N protein     can bind to = NO TRANSLATION

Question 17

Activate state –> Repressed state

Answer 17

• gene transcribed by RNAP
• RNA info converted into mRNA
• mRNA binds to AGO
• AGO with mRNA finds transcript and recruits enzymes important for regulating chromatin structure
• SILENCES CHROMATIN

Question 17

New definition of epigenetic:

Answer 17

gene expression changes that are mutation independent and heritable in the absence of the triggering event

Question 17

paramutation

Answer 17

an allele of a gene can permanently and heritably change the expression or behavior of another allele of the same gene, even without any changes in the DNA sequence

Question 17

Paramutation in corn

Answer 17

the presence of B’ alters B-I
- B-I that has been exposed to B’ can now alter B-I alleles

*NON-MENDELIAN INHERITENCE**
- inherited in a transgenerational manner

• expression of b1 locus of B-I allele is regulated by a B-I enhancer (Tandem repeats) approx. 1 kb 5’ from b1 locus
  
  • in the B’ locus, tandem repeats are associated with B’ silent chromatin

Question 17

RNAe (RNA epigenetics)

Answer 17

involves use of miRNA to recruit protein involved in modification of chromatin to specific genes

Question 17

A. thaliana (plants) RNAe

Answer 17

- uses RNAP 4 and 5 1. RNAP 4 transcribes region of genome 2. bound by RNA-dependent RNAP and a DS copy made 3. transcript diced 4. 20b miRNA sequences produces 5. AGO recognizes miRNA and forms complex 6. complex enters nucleus 7. Pol 5 transcribes region and recruits methylases 8. area of DNA is methylates = SILENT STATE

Question 17

S. pombe (yeast) RNAE

Answer 17

1. area of heterochromatin induces transcripts (using Pol2) 2. transcripts bound by RNA-dependent RNAP 3. copy of RNA made 4. copy recognized by dicer 5. 20 base long nucleotides produced 6. AGO recognizes 20b seq. and finds binding site and forms RNA scaffold 7. enzymes to modify histones are recruited = REPRESSIVE STATE (HETEROCHORMATIN MAINTAINED)

Question 17

C. elegans (nematode)

Answer 17

1. RNAs diced 2. placed in worm-specific AGO protein 3. enters nucleus and finds target transcript 4. recruit enzymes to modify chromatin structure = REPRESSIVE STATE (HETEROCHORMATIN MAINTAINED)

Question 17

Tandem repeats RNAe

Answer 17

when tandem repeats are transcribed, siRNA are produced which are recognized by AGO and enters the nucleus again where it binds to transcript produced from tandem repeats - Repression enzymes are brought in which change the structure of TR from open to silent and now TR cannot act as an enhancer

Question 18

mop

Answer 18

- characterization of genes required in trans - mop recognizes Pol4 and produces a RNA copy of transcripts, DS RNAs are then diced, associate with AGO and re-enter nuc to maintain silent chromatin

Question 19

transgenerational silencing in the germ line can last for ______ generations

Answer 19

>10

Question 20

transposable element gene silencing

Answer 20

cause massive gene rearrangements - activation of P element through transposition in flies leads to sterile phenotype

Question 21

Piwi pathway --> Piwi RNA expression

Answer 21

P-element Induced Wimpy testis - surpresses expression of transposable element transposons - pre-cursor piRNA transcript transcribed using RNAPII - Rhino protein shuttles between chromatin and RNA - when Rhino on RNA, it marks transcripts to be taken into cytoplasm where it is cleaved

Question 22

Piwi RNA function

Answer 22

- short RNA seq that piwiAGO recognizes , moves into nucleus - piwiAGO RNA complex will hybridize to transposon RNA = silencing of TE transcription and degradation of transposon mRNA - therefore NO TRANSPOSONS ARE BEING PRODUCED

Question 23

Piwi conversion

Answer 23

- new TE can be used to produce loci that will produce PiwiRNA Transposon RNA --> piwiRNA locus (suppress expression of transposon)

Question 24

transgeneration avoidance of Pseudomonas

Answer 24

- C.elegans originally attracted to pseudomonas (pathogenic) - however once on pseudomonas, c. elegans learn to avoid it which can last for many generations - learning index maintained for 4 generations, lost in the fifth *REQUIRES USE OF miRNA*

Question 25

P0 HK9me3 methyltransferase

Answer 25

methylates histones and loses the organisms' ability to learn

Question 26

Prions

Answer 26

- protein disease, infectious agent non prion (has same aa seq as prion) -> exposed to prion protein --> changes structure of protein to become --> prion **PERMITS TRANSGENERATIONAL CHANGE IN PROTEIN CONFORMATION**

Question 27

mot3

Answer 27

TF mot3- = suppresses expression MOT3+ (prion form) = INACTIVE, allowing expression - MOT3+ is stuck in an aggregate such allows expression of gene - MOT3+ can convert mot3- to MOT3+ for generations