Bio112 Exam 2 part 2

Sense strand vs Antisense strand DNA

Anti-sense - template for mRNA sense strand

Sense strands - non coding strand of DNA

Guo and Kemphus C elegans embyro experiment

Wanted to figure out the effects of different RNAs on gene expression

Injected par-1 antisense into the CE embryo using another sample with the sense strand as a negative control

Found antisense to cause embryonic lethality (meaning par-1 is important to embryonic development) 

Surprise (Found that the sense strand also had a similar effect)

Andrew Fire, Craig Mello findings about RNAi

Wanted to figure out the effects of Diff RNA types on gene expression

Discovered RNAi

+Found that dsDNA was most effective at interference

→only a few molecules were nessasary do there had to be an Amplification mechinism.

SiRNA mechanism

dsRNA cleaved by DICER (both endonuclease III RNase and RNA helicase) into siRNA

RISC complex brings siRNA and targets the mRNA strand and cleaves alongside Argonaut, which does the actual degradation.

SiRNA Amplification mechinism (also parts of dicer)

RdRP(Rna Dependent RNA polymerase) makes a complimentary with siRNA and an mRNA strand to amplifiy the signal and start the cycle again

RNAi is present in Eukaryotes or Prokaryotes?

Eukaryotes (prokaryotes have Crispr)

Argonautes

Catalytic component of RISC →endonucelase activity again mRNA that RISC is bound to

Functions of RNAi

1. Defense agaginst foreign material (viral RNA)

2. Removes unwanted/damaged RNA →siRNA pathway

3. Down regulatio of gene expression

4. Maintaining genome stability

miRNA

Encoded by the genome

Directs post-transcriptional repression

regulates mRNA expression in humans and mammals,

Biogensis of miRNA

Starts as Pri-miRna stem loop shaped in nucleus

Drosha releases the stem loop creating

Pre-miRNA → hairpin-shaped, which is exported from the cell

Dicer cuts of hairpin loop, making it into a mature miRNA duplex

delivered to RISC and inhibits or degrades mRNA based on sequence matching.

How does RNAi down regulate gene expression?

Centromeric domains are heterochromatin→

RNAi helps in H3K9-methylation bound by HP1

low acetylaion and not very expressed due to compaction

Safeguarding Heterochromatin domains (gene expression RNAi)

Transcription of heterochromatin domains leads to dsRNA

• activates RNAi

dsRNA→dicer→siRNA→RISC/Ago→RdRP for signal amp

This process also recruits H3K9 Methylase and restores hetero chromatin domain and silencing gene expression

piRNA (maintaining genome stability)

-Silences transposable elements

• binds to PIWI protein and protects germline from transposable elemnts during gamete meisos

TEs may be activated due to demethylation of DNA but piRNA binds ot pwi protein to target and silence TEs(using slicer post transcriptionally) and remethylate DNA (pretranscriptional)

Pre-transcriptional Silencing by siRNA

Directs DNA methylation and heterocrhomatin silencing

Elements present for the formation of life

NH3, CH4, H2, H2O, H2S, HCN

Importance of 2’OH on RNA

Very reactive → directs self hydrolysis catalyzed

• 2’OH can act as a nucleophile under neutral and basic conditions.

Products of translation of ssRNA

form ds Regions

clsssify RNA based on structure

Most RNA is processed by Spliceosomes

Phosphodiester bond is cut and rejoined

Self splicing RNA (and the scientists) mechinism and significance 

Catalytic properties of RNA (Altman and Cech)

• This means RNA can possible act as both the template and catalyst creating a new possible origin of life.

• RNA has been found also to self replicate

Properties of enzymes

Catalyzes reactions, specific reactions, is neither altered nor consumed, multiple turnovers.

Ribozymes

RNA that acts as an enzyme (peptide bond formation, ester bond cleavage, splicing, ligations)

RIbosomes compisition 

Mostly RNA →active site is a ribozyme

RNA world hypothesis

RNA can act as both the storer of genetic information and enzymes

RNa can refualte itself and other processes

can catalyze reactions like enzymes

Could have been origin of life

Biological orgins of life

Biological molecules can arise from abiotic sources, RNA can serve as both catalyst an template,

DNA took over genetic role,

Proteins took over most catalytic roles.

Some key mechanisms, such as protein synthesis, are still governed by RNA

RNA world pathway

Formation of earth→hydrosphere→Prebiotic chemistry(Uruy-miller) →Pre-RNA world (nucleotides and organic molecules) → RNA world→First DNA/Protein life→Diversification of life