Molec BIO Exam 3: Deck 1/lec 1

Need to study this!!!!

What are small regulatory RNA’s? 1What do they do, 2how big are they, 3are they translated, 4what organisms are they found in, 5what are some examples of the different types of small regulatory RNA’s

1: They influence gene expression and other cellular processes.

2: They are typically less than 200 nucleotides in length

3: They are not translated/they are non-coding

4; They are found in all organisms, ubiquitous(present in all places)

5: There are siRNAs, miRNAs, piRNAs ect.

What were the 3 specific early clues for the RNAi pathway?

1: RNA interference(RNAi); the process by which ds-RNA induces gene silencing.

2: Gene silencing: genes are not expressed via blocking transcription and or post-transcriptional events.

3: RNAi is usually a post- transcriptional even that affects RNA stability and or translation

What was another big clue about the RNAi pathway(Nobel prize)?

1. Unexpected gene silencing in plants (1990): Researchers attempting to increase pigment production in petunias by introducing additional pigment-producing genes instead observed reduced pigmentation. This phenomenon became known as co-suppression.

2. Antisense RNA experiments: Scientists discovered that introducing antisense RNA (complementary to an mRNA) could inhibit gene expression. However, sometimes sense RNA also produced gene silencing, which was unexpected.

3. The Fire and Mello experiment (1998): Researchers tested sense RNA, antisense RNA, and double-stranded RNA in C. elegans. They discovered that dsRNA was dramatically more effective at silencing genes than either single-stranded RNA alone.

4. Sequence specificity: The dsRNA only silenced genes with complementary sequences, suggesting a targeted mechanism rather than general RNA degradation.

What made Fire and Mello's discovery so important?

Conceptual comparison of the RNA treatments in the original experiments.

The surprising finding was that dsRNA, not single-stranded RNA, was the particularly powerful trigger for gene silencing.

This eventually helped scientists identify the molecular machinery of RNAi, including Dicer, siRNA, and RISC.

What was another early clue for the RNAi pathway in relation to Mex-3-mRNA(in situ hybridization?

When looking at 4 Petri dishes:

A has no Mex 3 staining

B has mex3 staining

C; has antisense mex3 staining

D; has ds-RNA mex-3 and mex-3 stain.

Results:

A: No stain as expected( neg control)

B: Staining is present as expected( pos control)

C: Staining is present( a little lighter then the straight up mex3 stain)

D; no stain present at all(important)

Conclusion; There seems to be some sort of regulatory mechanism that is present on DS-RNA when compared to sense and anti-sense RNA( sense= single stranded RNA(5’→3’), anti-sense RNA=( 3’→5’) single stranded RNA, Ds RNA has both sense and anti sense strand( sense on top and anti-sense on bottom)

Why was this slide another early clue about RNAi pathway?

It shows that the effect is post-transcriptional because it has the effect on RNA and not DNA , therefore this pathway has to be post transcriptional given that it acts on RNA that is made during transcription.

Also this slide shows that RNAi has sequence specificity, it has specific effects on specific genes: not the same defined effect on all genes.

This slide also shows us that the dsRNA is potent: it produces big effects that point to this, not just making minor changes.

What is the molecular mechanism of RNAi? Define several RNAi terms: Dicer, argonaute(slicer), RNA-induced-silencing complex(RISC), siRNA, guide strand and passenger strand.

Dicer: enzyme that cleaves ds-RNAs into siRNAs

Argonaute(slicer): enzyme that cleaves mRNA using siRNA as a guide

RISC: complex of Argonaute and siRNA

siRNA: short interfering RNA

Guide strand: siRNA strand associated with RISC

passenger strand: siRNA strand removed from RISC(going for a ride rather than stay in the complex).

What are the two types of dsRNA sources for RNAi pathway?

Endogenous(Internal): Naturally occur in the host cell.

Exogenous(External):dsRNA delivered to cell( ex: by virus or investigator)

What is the cytoplasmic RNAi pathway in C-elegans(Steps)?

Whole number for result, 0.5 for intermediate

0: Starts with dsRNA

0.5: Dicer/RDE-4 added

1: siRNA

1.5: Argonaute and additional proteins added

2: RISC— SiRNA integrated

2.5; RISC recognizes mRNA

3: Gene Silencing

Answer:

Passenger:5’GGGUUAACGACACGUCUCU3’

Guide. :5’CCCAAUUGCUGUGCAGAGA3’

How does RNAi act as an immune system( What kind of protection does it provide)

RNAi provides viral protection.

How does RNAi act as an immune system( Which cellular RNAs are normally present as siRNAs)?

1. Retrotransposons: Mobile genetic elements that can disrupt DNA; RNAi silences their RNA to protect the genome.

2. INGI (18%): Retrotransposon family whose RNA produces ~18% of observed siRNAs, demonstrating RNAi defense.

3. SLACS (13%): Another retrotransposon family producing ~13% of observed siRNAs, demonstrating RNAi defense.

Key takeaway: RNAi acts as a genomic immune system by silencing retrotransposons and limiting harmful DNA insertions.

How does RNAi act as an immune system( in terms of Retrotransposon RNAs are cleaved by the RNAi machinery)

Retrotransposons are jumping genes that require an RNA intermediate

Retrotransposons produce dsRNA, which Dicer cleaves into siRNAs. These guide RISC to degrade retrotransposon RNA, preventing its replication and protecting the genome. So retrotransposons play into the function that has RNAi has.Retrotransposons are bad but RNAi is right there to regulate it(complementary relationship almost)

1. Retrotransposons are jumping genes that can disrupt DNA.

2. They produce dsRNA, which triggers the RNAi pathway.

3. RNAi destroys their RNA, preventing them from making additional copies.

How does RNAi regulate gene expression(Q: Can cells use RNAi to influence expression of cellular mRNAs?)

A: Yes. Cells produce miRNAs that guide RISC to complementary cellular mRNAs, inhibiting translation or promoting mRNA degradation.

Key distinction: Dicer does not need to cut the cellular mRNA itself. It processes the miRNA, which then guides RISC to regulate the mRNA.

This is how RNAi can function in both defense against foreign or harmful RNA and normal regulation of cellular gene expression.

MicroRNAs(miRNAs)→ Definition, compare to siRNAs(general/is it the same), what type of organisms have miRNAs, how many miRNA genes do humans have, what kind of base pairing to mRNA?

miRNAs: small siRNA-like RNAs that alter gene expression

miRNA is similar to siRNAs in some ways and different in others

Found in many eukaryotes

Humans have 1,000 miRNA genes

Imperfect base pairing to mRNA

What are the terms for miRNA pathway?

Pri-miRNA: the primary transcript generated by transcription

Drosha: Cleave pri-miRNA to generate pre-miRNA(Dicer substrate)

Dicer: cleaves pri-miRNA to generate miRNA

RISC: same as RISC before, not necessarily the same as siRNA but can be.

What are the steps for miRNA formation both specifically in the nucleus and outside the nucleus.

Back of Flashcard: miRNA Formation

1. RNA Polymerase II transcribes DNA into pri-miRNA (contains hairpin structures).

2. Nucleus – Two pathways:

• Canonical: Drosha + Pasha cleave pri-miRNA into pre-miRNA.

• Alternative (Mirtrons): RNA splicing + lariat debranching produce pre-miRNA, bypassing Drosha/Pasha.

3. Exportin-5 (EXP-5) transports pre-miRNA from the nucleus into the cytoplasm.

4. Dicer (DCR-1) + LOQS cleave pre-miRNA into a short miRNA duplex (~22 nucleotides).

5. AGO1-RISC retains the guide strand and removes the passenger strand.

6. Final Function: miRNA guides RISC to complementary cellular mRNA, inhibiting translation and promoting mRNA degradation.

Key takeaway: Both nuclear pathways produce pre-miRNA, which enters the cytoplasm and is processed by Dicer → RISC to regulate cellular gene expression.

How does miRNA mature in the cytoplasm, and how does it regulate gene expression?

Back: miRNA Maturation in the Cytoplasm

1. Dicer (DCR-1) + LOQS cleave pre-miRNA into a short miRNA/miRNA* duplex (~22 nucleotides).

2. Loading complex transfers the miRNA duplex into AGO1-containing pre-RISC.

3. Strand selection: The passenger strand (miRNA*) is removed, while the guide strand (miRNA) is retained.

4. Mature AGO1-RISC uses the guide strand to recognize complementary sequences on target mRNA.

5. Final function: RISC inhibits translation and promotes mRNA destabilization/degradation, reducing gene expression.

Key takeaway: Dicer processes pre-miRNA → AGO1-RISC retains the guide strand → miRNA guides RISC to suppress cellular mRNA expression.

How does miRNA-RISC (miRISC) regulate gene expression after recognizing a target mRNA?

miRNA Mechanism of Action

1. mRNA Recognition: miRNA guides RISC to complementary sequences on target mRNA.

2. mRNA Cleavage & Degradation: miRISC can promote mRNA breakdown, preventing protein production.

3. Translation Inhibition: miRISC interferes with ribosome activity, preventing translation of mRNA into protein.

4. P-body Sequestration: Repressed mRNAs can accumulate in processing bodies (P-bodies), where they may be stored or degraded.

Key takeaway: miRNA-RISC reduces gene expression by promoting mRNA degradation or inhibiting translation, sometimes involving P-bodies.

Important distinction: Direct Argonaute-mediated cleavage generally requires extensive miRNA–mRNA complementarity. Most animal miRNAs instead promote translational repression and mRNA destabilization.

What are the six proposed models for how miRNA-RISC inhibits gene expression?

Models for miRNA Action

1. Translation Elongation Inhibition: RISC interferes with ribosomes moving along mRNA, preventing protein synthesis.

2. Co-translational Protein Degradation: Newly synthesized proteins are degraded during translation.

3. Competition for 5′ Cap: RISC interferes with translation initiation factors (eIF4E), preventing ribosome recruitment.

4. Inhibition of Ribosomal Subunit Joining: RISC prevents proper assembly of ribosomal subunits, blocking translation initiation.

5. Inhibition of mRNA Circularization: RISC promotes poly-A tail shortening (deadenylation), disrupting interactions between the 5′ cap and 3′ tail that support translation.

6. Deadenylation & Decapping: RISC promotes removal of the poly-A tail and 5′ cap, leading to mRNA degradation.

Key takeaway: miRNA can reduce protein production by blocking translation, promoting protein degradation, or destabilizing mRNA.

Note: These are proposed mechanistic models from the slide, not necessarily six equally established or independent pathways. Deadenylation and subsequent mRNA decay are particularly well-supported mechanisms.

Compare and contrast the siRNA and miRNA

Similarities:

• Both form double-stranded RNA duplexes containing a guide strand and passenger strand.

• Both have short 3′ overhangs.

• Both undergo cleavage and loading into Argonaute-containing RISC.

• Both regulate gene expression.

Differences:

Feature	miRNA	siRNA
RNA structure	Hairpin with mismatches/bulges	Long, nearly perfectly paired dsRNA
Strand pairing	Imperfect complementarity	Nearly perfect complementarity
Argonaute	miRNA-associated Argonaute	siRNA-associated Argonaute
Mechanism	Represses translation and promotes RNA decay	Cleaves target RNA

Key takeaway: miRNA has imperfect base pairing and primarily represses translation/promotes mRNA decay, while siRNA has nearly perfect base pairing and directs target RNA cleavage.

This combines the structural comparison from Slide 1 with the functional comparison from Slide 2, without adding mechanisms that aren't shown in your professor's slides.

How are small RNAs used for experimental and therapeutic purposes?(Laboratory specific(general)) How can you study gene function in the laboratory?

Isolate natural mutants

Overexposes gene

Create gene knockouts(remove or disrupt gene from genome)

create RNAi knockdowns

How are small RNAs used for experimental and therapeutic purposes? What are the RNAi knockdown approaches( they trick cells into destroying their own RNAs)

1; Introduce long, dsRNA into cells

2: Introducing siRNAs into cells

3: Introduce plasmid DNA with promoter to generate long dsRNA into cells(regulated promoter)

4: Introduce plasmid DNA with promoters to generate short hairpin RNAs(shRNA)that enters the RNAi pathway(regulated promoter)

Approach	What enters the cell?	What happens next?
1	Long dsRNA	Dicer → siRNA → RISC
2	siRNA	RISC loading
3	Plasmid DNA	Long dsRNA produced → Dicer → RISC
4	Plasmid DNA	shRNA produced → processing → RISC

What therapies include RNAi?