Molecular

Cellular

Biology-I

(MCB-I)

PCB 6025

M. Alejandro Barbieri

Office: HLS 318C/214

Hours: by appointment.

Email: barbieri@fiu.edu

1-Regulatory RNA, 2-RNA interference and micro RNA, 3-Retroviruses, 4-Transposons and Retroposons, 5-Promoters and Enhancers, 6-Activating Transcription, 7-RNA Splicing and Processing, 8-Chromosomes-Nucleosomes, 9-Controlling Chromatin Remodeling and Structure.

TOPICS-I

10-Gene Regulation I, 11-Gene Regulation II, 12-Protein Synthesis

TOPICS-II

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Books

Molecular Biology of the Cell

Genes IX/X/...

Any....

5

Presentation: Topic selection/student

Regulatory RNA (review and paper)

RNA interference

Micro RNA

Transposons, Retroposons and Retroviruses

Promoters and Enhancers

Activating Transcription

RNA Splicing and Processing

Controlling Chromatin Structure and Chromatin remodeling

Retroviruses

Gene Regulation

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Evaluation:1- Exam= 50% (Parts-I and II)

2-Presentation 1=50%* (review and specific paper, idea, concept and experimental-selected by the instructor)

2a-presentation: 40%2b-participation: 10%

(*)-Each student will make a presentation for 15 min. (for specific papers) and 20 min (for review papers) with discussion (2-3 questions).

Exam

(Topics plus selected papers)

Open/Close book

Regulatory RNA

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MicroRNAs Are Regulators in Many Eukaryotes

• Animal and plant genomes code for many short (∼22 base) RNA molecules called microRNAs.

• MicroRNAs regulate gene expression by base pairing with complementary sequences in target mRNAs.

C. elegans: regulator gene lin4 and its target gene lin14

(lin: Proteins for larval development)

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RNA Interference Is Related to Gene Silencing

• RNA interference triggers degradation of mRNAs complementary to either strand of a short dsRNA.

Figure 13.21

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• dsRNA may cause silencing of host genes.

Figure 13.22

What is RNA interference?

Shooting down mRNA

BackgroundBackgroundWhat is it?What is it?Why use it?Why use it?The mechanism and processThe mechanism and processExperimental considerationsExperimental considerations

RNAiRNAi

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Plasmid

Virus

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Jorgensen 1990van der Krol 1990

Gene injection (pigmentationEnzyme-petunias)Expectation: more red colorCo-suppression of transgene and endogenous gene.

Bill Douherty and Lindbo 1993Gene injection with a complete tobaccoetch virus particle.Expectation: virus expressionCo-suppression of transgene and virus particles via RNA.

Hamilton and Baulcombe 1998Identification of short antisense RNA sequences dsRNA?How?

Fire and Mello 1998Injection of dsRNA into C. elegansRNA interference (RNAi) or silencing

Ambros 1993 (2000)Identification of small RNA in C. elegans (micro RNA)

18Shooting mRNA means RNA interference

What is RNA interference?

--Gene “knockdown”

--A cellular mechanism that degrades unwanted RNAs in the cytoplasm but not in the nucleus. Why?

--A way for the cell to defend itself.

Why use RNAi?

1. The most powerful way to inhibit gene expression and acquire info about the gene’s function fast

2. Works in any cell/organism

3. Uses conserved endogenous machinery

4. Potent at low concentrations

5. Highly specific.

The mechanism of small interfering RNAs (siRNAs)

What happens?

dsRNA is processed into shorter units (siRNAs) that guide the targeted cleavage of homologous

RNA.

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RNA interference:

--A type of gene regulation

--Involve small RNA molecules

--Induce a double stranded RNA

The RNAi process

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Step 1

• dsRNA is processed into sense and antisense RNAs • 21-25 nucleotides in

length • have 2-3 nt 3’

overhanging ends • Done by Dicer (an

RNase III-type enzyme)

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Step 2

• The siRNAs associate

with RISC (RNA-

induced silencing

complex) and

unwind

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Step 3

• the antisense siRNAs act as guides for RISC to associate with complimentary single-stranded mRNAs.

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Step 4

• RISC cuts the mRNA approximately in the middle of the region paired with the siRNA

• The mRNA is degraded further

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Catalysis: RdRP copies RNA making more ds RNA.

Dicer complex: RNAase III with ATP hydrolysis requirement.Dicer cuts, unwinds dsRNAand generates more siRNA.

More RdRP is activated and more dsRNA is made.

RISC complex:RNA-InducibleSilencing Complex with ATPhydrolysis.

ssRNA (exogenous)RNA-dependentRNA polymerase

(endogenous)

(RdRP)

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Gene regulation by small RNAs

Small temporal (St) RNAs prevent translation to stop gene expression quickly

siRNAs degrade mRNAto stop gene expression quickly

Dicer gene in C. elegans

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--MicroRNAs (miRNA) are single-stranded RNA molecules of about 21-23 nucleotides in length, which regulate gene expression (down-regulation). --miRNAs are encoded by genes that are non-coding RNAs ( no proteins are made)--Stem-loop or hairpin loop intra-molecular base pairing is a pattern that can occur in single-stranded DNA or, more commonly, in RNA.

-your RNAi?

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Experimental Considerations

Transfection method: 1-Lipofectamine 2000--cationic lipids to bind siRNA and neutral

lipids to allow escape from Endosomes

2-Plamids/Viruses--express small fragment of hairpin DNA

Transfection efficiency

Negative controls --scrambled siRNA

Off-target effects:

Sense (or antisense) strand is homologous to another sequence

Activation of stress response pathways “apoptosis”

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http://www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.htmlhttp://www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.html

Growth Factor Receptor Binding Protein (Grb) 2-mediatedRecruitment of the RING Domain of Cbl to the EpidermalGrowth Factor Receptor (EGFR) Is Essential and Sufficient toSupport Receptor Endocytosis

Fangtian Huang and Alexander Sorkin

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Knockdown of growth factor receptor binding protein 2 (Grb2) by RNA interference strongly inhibits clathrin-mediatedendocytosis of the epidermal growth factor receptor (EGFR).

To gain insights into the function of Grb2 in EGFRendocytosis, we have generated cell lines in which endogenous Grb2 was replaced by a modified yellow fluorescent protein(YFP)-tagged Grb2 and it was expressed at the physiological level.

In these cells, Grb2-YFP fully reversed the inhibitory effect ofGrb2 knockdown on EGFR endocytosis and, moreover, trafficked together with EGFR during endocytosis.

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To generate HeLa cells stably expressing Grb2-YFP, endogenousGrb2 was knocked down using vector-based shorthairpin RNA (shRNA) with simultaneous expression ofGrb2-YFP that has silencing mutations rendering this constructinsensitive to shRNA.

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pSilencer1.0-U6 vector--pSuper-H1 vector--

Type III RNA pol III promoter ----U6 small nuclear promoter (U6) ----human H1 promoter (H1)

retrovirus//inducible