Exam 4 part 1 Flashcards Preview

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Flashcards in Exam 4 part 1 Deck (29):
1

RNAi

the pathway where specific genes are inhibited
short RNA oligonucleotides in a protein complex regulate gene expression

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RNA oligonucleotide

targets protein complex to the desired RNA

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protein complex

inhibits gene expression

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RNA component of RNAi pathway

a short single strand of RNA that provides specificity

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protein component of RNAi pathway

made up of a collection of nucleic acid binding proteins, nucleases, and other enzymes that interact with RNA to regulate gene activity

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exogenous source of RNAi

RNA viruses or experiments
siRNA- short-interfering RNA

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siRNA

1. mostly exogenous
2. dsRNA precursors
3. may be target specific
4. degrade the target RNA

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endogenous source of RNAi

miRNA

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miRNA

1. encoded by endogenous genes
2. hairpin precursors
3. recognize multiple targets
4. can trigger transcriptional inhibition or mRNA cleavage

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RNAi two phase process

1. initiation- generation of mature siRNA or miRNA
2. execution- silencing of target gene, degradation or inhibition of translation

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initiation

1. cropping
2. dicing
3. activation

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cropping

1. occurs in nucleus
2. made of Pasha and Drosha "crop" pri-miRNA to form pre-miRNA
3. exported to cytoplasm
4. pre-miRNA has a 5' phosphate and a two-nucleotide 3' overhang

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Pasha

dsRNA binding protein

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Drosha

RNase III (cleaves dsRNA)

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dicing

1. occurs in cytoplasm
2. Dicer binds and dices pre-miRNA to form short dsRNAs
3. dicer has a "ruler" built in, cuts dsRNAs the same length
4. dicer cleaves ~22 nucleotide RNA duplex with 2bp 3' overhang

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activation

1. process of combining the short miRNA with effector proteins to form complex
2. generates mature RNA-induced silencing complexes (RISCs)

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Key members of RISCs

argonautes
humans have 4 argonauts proteins
C. elegans have 29

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RISC formation

1. dsRNA generated by dicer is bound by RISC-loading protein
2. once loaded Argonaute (Ago2) selects one strand of dsRNA
3. this is guide strand, other "passenger" strand is either degraded or removed

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guide strand

retained in holo-RISC

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RNAi targeting

miRNA guide strand has a seed sequence at the 5' end that is complementary to the target mRNA

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inhibition

if miRNA is complementary, Argonaute cleaves the mRNA
incomplete complementarity, translation is inhibited

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RNAi useful

can silence a gene without creating a mutant

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HCV infection

can only infect cells that express miR-122
silencing miR-122 represses viral infection

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CRISPRs

Clustered Regularly Interspaced Short Palindromic Repeats

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CRISPR adaptation

short pieces of viral RNA get integrated into the bacterial CRISPR loci

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CRISPR crRNA biogenesis

CRISPR RNA is now synthesized (tracr RNA and guide RNA)

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CRISPR interference

interact with the protein (Cas9) and cleave the viral DNA

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tracr RNA

helps to activate Cas9

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gRNA (guide RNA)

helps to silence the target viral DNA