Molecular Biology Chapter 20: Genome Defense

Antisense RNA is complementary to which sequence?

Target sequence

Antisense RNA is how many bases long?

<150

Antisense RNA defense involves preventing what process?

Translation

Antisense recognizes which type of RNA as foreign and destroys it?

dsRNA

What is RNA interference (RNAi)

The introduction of double-stranded RNA into an organism to selectively knock-down gene expression. Sequence specific and destroys dsRNA and sRNA (like mRNAs)

Where does RNA interference (RNAi) occur

Eukaryotes

What triggers RNAi?

Viral dsRNA or transposons

1. dsRNA recognized by RDE-4 and proteins

2. Dicer cleaves dsRNA into 21-23 bp fragments with 2 nt overhangs

3. siRNA recruited into RISC (RNA-induced Silencing Complex)

4. RISC activated, strands separated --> ssRNA guide

5. Finds complementary RNA sequences

6. Perfect match -> RISC cleaves and degrades target

siRNA pathway

1. Pri-__RNA made in nucleus

2. Processed by Drosha -> pre-__RNA

3. Exported to cytoplasm

4. Trimmed by Dicer --> mature __RNA

5. Associates with __RISC

6. Imperfect match -> translation repression (not degradation)

miRNA pathway

From external dsRNA (Viruses, transposons)

Perfect complementarity -> RNA degradation.

siRNA

Made by cell (pri to pre) for gene regulation

Imperfect complementarity -> translation repression

miRNA

Differences between miRNA and siRNA include:

From external dsRNA (Viruses, transposons)

Perfect complementarity -> RNA degradation.

Made by cell (pri-miRNA -> pre-miRNA)

Imperfect complementarity -> translation repression

True

piRNAs are known as what?

Piwi-Interacting RNAs

piRNAs (piwi-interacting RNAs) protect germline DNA, cluster in repeats near centromeres, and are modified at 3' end.

True

What enzyme consists of a dsRNA-binding domain

PAZ domain (binds 3' overhang)

Two RNase III domains (cut on opposite sides)

Creates 21-23 nt fragments.

Dicer

Protein that is part of RISC thatr binds to both the 3' and 5' ends strand of the miRNA or siRNA molecule.

Argonaute protein

Amplifies RNAi effect

Uses cleaved target as template -> more dsRNA

More dsRNA cleaved by Dicer -> more siRNAs

Can spread cell-to-cell (C. elegans: across generations) (not seen in mammals)

RdRP (RNA-dependent RNA polymerase)

How can siRNAs be used as a Drug?

Can inject dsRNA into cell or put construct into cells, produces dsRNA in vivo that are then cut by Dicer. Introducing into cell decreases expression of gene.

Experimental Induction of RNA Interference involves:

Sense/antisense hairpin

Double promoter

Two genes, sense and antisense

True

What are the challenges of using siRNA therapeutically?

Stability of siRNA, Delivery method lipids and nanoparticles still can have off-target effects.

CRISPR stands for?

Clustered Regularly Interspaced Short Palindromic Repeats

The three stages of CRISPR are:

Adaptation, Expression, Interference

What stage of CRISPR is the following?

Viral sequences incorporated into CRISPR locus near PAMs

(protospacer adjacent motif (or PAM for short) is a short DNA sequence (usually 2-6 base pairs in length) that follows the DNA region targeted for cleavage by the CRISPR system)

Adapation

What stage of CRISPR is the following?

CRISPR locus transcribed and processed into crRNAs (Circulating RNAs)

Expression

What stage of CRISPR is the following:

crRNAs + Cas proteins recognize and degrade foreign nucleic acids

Interference

Spacers (memories) separated by repeats

CRISPR array

Store or degrade sequences

Cas proteins

PAMs (Protospacer Adjacent Motifs) 2-6bp sequences on target

True

CRISPR Classifications

Class 1: Multi-protein effector complexes

Type I: Targets dsDNA, uses Cas3 nuclease, requires PAM

Cas3 = helicase + nuclease, makes single-strand breaks

Type III: Targets transcribing sequences (RNA + DNA)

No PAM required

Cas7 cuts RNA, Cas10 cuts DNA simultaneously

Class 2: Single effector protein

Type II: Most common for genome editing

Components: Cas9, tracrRNA, crRNA

tracrRNA + RNase III + Cas9 process pre-crRNA

crRNA + tracrRNA + Cas9 recognize target

Creates double-strand breaks with blunt ends

(no answer)

Combines crRNA and tracrRNA into one molecule

Simplifies delivery (one molecule instead of two)

Lab-Made sgRNA, which stands for?

Single Guide RNA

Genome Editing Applications

DNA Repair After Double-Strand Breaks

1. NHEJ (Non-Homologous End Joining)

No sequence homology required

Easier for insertions/deletions

Less predictable, more off-target effects

2. HDR (Homology-Directed Repair)

Requires sequence homology

More predictable

Fewer off-target effects

Can insert specific sequences

dCas9 (Dead Cas9)

No nuclease activity but still binds DNA

Applications:

Block RNA polymerase → repress transcription

Fused to transcription factors → activate transcription

Tissue-specific gene expression (with UAS/Gal4 system)

Medical Applications of genome editing include:

Fighting antibiotic-resistant bacteria (delivered via bacteriophage)

Targeting antibiotic resistance genes on plasmids

True

(Other genome editing tools)

Zinc fingers recognize 3-4 nucleotides each

Fused to FokI endonuclease

Requires dimerization for activity

Expensive but no PAM needed

Fewer off-target effects

Zinc Finger Nucleases

(Other genome editing tools)

TALE domain: 33-35 aa repeats

Two amino acids determine base recognition (RVDs)

NI = A, NG = T, NN = G, HD = C

Fused to FokI

Recognizes 16-24 bp sequences

Transcription Activator-Like Effector Nucleases

(Other genome editing tools)

Modified amino acid backbone (not sugar-phosphate)

Form triplex with DNA (PNA/DNA/PNA)

No nucleases

Repaired by HDR or nucleotide excision repair

PNAs (Peptide Nucleic Acids)