Week 11 - micro RNA and RNA-epigenetic Flashcards
Heterochronic genes
mutants affect timing of divisions
lin-14 (lf)
lineage is disrupted; precocious development of L2 lineage
- 2 rounds of division in L1 larval stage
lin-14 (gf)
suppression of L2 lineage and replacement with L1 lineage
- continuous reiteration of L1 lineage
lin-4 (lf)
- result in same suppression of L2 lineage as Lin-14 gf
- continuous reiteration of L1 lineage
Double mutants;
lin-4 (lf) lin-14 (lf)
demonstrated lin-14 phenotype
Lin-4 ____ lin-14
represses
lin-14 encodes a _______
nuclear protein
lin-4 encodes a _______
non-coding RNA, a miRNA the first found
lin-4 RNA ____ with Lin-14 _______
base pairs, 3’ UTR
in lin-14 gf mutants, ________ are deleted
lin-4 binding sites
lin-14 mRNA is expressed ______ larval stages, however lin-14 protein is expressed in _____ larval stages
throughout, L1
lin-4 mRNA is expressed at ______ larval stages
L2, L3 and L4
Let-7 and Lin-29
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
let-7 encodes ____ and can bp at the _____ of RNA
non-coding RNA, 3’ UTR
miRNAs
- everywhere
- very short; 20 odd bases long
- sequencing 20 base RNAs uncovered many miRNAs
miRNA transcription
- 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
miRNA suppresses ________
translation
RNAi degrade _____ through _____
mRNA, cleavage
miRNA guide ____ to ____ and ___ suppresses _____
RISC,
specific mRNAs,
RISC,
translation
(MODEL SUGGESSTS miRNA HAVE NO ROLE IN ACTIVATING RISC)
Experiment to understand miRNA role in RISC proteins
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
Activate state –> Repressed state
- 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
New definition of epigenetic:
gene expression changes that are mutation independent and heritable in the absence of the triggering event
paramutation
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
Paramutation in corn
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
RNAe (RNA epigenetics)
involves use of miRNA to recruit protein involved in modification of chromatin to specific genes
A. thaliana (plants) RNAe
- uses RNAP 4 and 5
- RNAP 4 transcribes region of genome
- bound by RNA-dependent RNAP and a DS copy made
- transcript diced
- 20b miRNA sequences produces
- AGO recognizes miRNA and forms complex
- complex enters nucleus
- Pol 5 transcribes region and recruits methylases
- area of DNA is methylates
= SILENT STATE
S. pombe (yeast) RNAE
- area of heterochromatin induces transcripts (using Pol2)
- transcripts bound by RNA-dependent RNAP
- copy of RNA made
- copy recognized by dicer
- 20 base long nucleotides produced
- AGO recognizes 20b seq. and finds binding site and forms RNA scaffold
- enzymes to modify histones are recruited
= REPRESSIVE STATE (HETEROCHORMATIN MAINTAINED)
C. elegans (nematode)
- RNAs diced
- placed in worm-specific AGO protein
- enters nucleus and finds target transcript
- recruit enzymes to modify chromatin structure
= REPRESSIVE STATE (HETEROCHORMATIN MAINTAINED)
Tandem repeats RNAe
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
mop
- 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
transgenerational silencing in the germ line can last for ______ generations
> 10
transposable element gene silencing
cause massive gene rearrangements
- activation of P element through transposition in flies leads to sterile phenotype
Piwi pathway –> Piwi RNA expression
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
Piwi RNA function
- 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
Piwi conversion
- new TE can be used to produce loci that will produce PiwiRNA
Transposon RNA –> piwiRNA locus (suppress expression of transposon)
transgeneration avoidance of Pseudomonas
- 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
P0 HK9me3 methyltransferase
methylates histones and loses the organisms’ ability to learn
Prions
- 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
mot3
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
