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TOPICS-I. 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.

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TOPICS-I

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-II

10-Gene Regulation I,

11-Gene Regulation II,

12-Protein Synthesis


Books

Molecular Biology of the Cell

Genes IX/X/...

Any....


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


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



Micrornas are regulators in many eukaryotes
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)


Rna interference is related to gene silencing
RNA Interference Is Related to Gene Silencing

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

Figure 13.21



What is RNA interference?

Shooting down mRNA


RNAi

  • Background

  • What is it?

  • Why use it?

  • The mechanism and process

  • Experimental considerations


Plasmid

Virus


Jorgensen 1990

van der Krol 1990

Gene injection (pigmentation

Enzyme-petunias)

Expectation: more red color

Co-suppression of transgene

and endogenous gene.

Hamilton and Baulcombe 1998

Bill Douherty and Lindbo 1993

Identification of short antisense RNA

sequences

dsRNA?

How?

Gene injection with a complete tobacco

etch virus particle.

Expectation: virus expression

Co-suppression of transgene

and virus particles via RNA.

Ambros 1993 (2000)

Fire and Mello 1998

Identification of small RNA in

C. elegans (micro RNA)

Injection of dsRNA into C. elegans

RNA interference (RNAi) or silencing


Shooting 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

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.


The rnai process
The RNAi process

RNA interference:

--A type of gene regulation

--Involve small RNA molecules

--Induce a double stranded RNA


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)


Step 2

The siRNAs associate

with RISC (RNA-

induced silencing

complex) and

unwind

Step 2


Step 3

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

Step 3


Step 4

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

The mRNA is degraded further

Step 4


ssRNA (exogenous)

RNA-dependent

RNA polymerase

Catalysis: RdRP copies

RNA making more ds RNA.

Dicer complex: RNAase III

with ATP hydrolysis

requirement.

Dicer cuts, unwinds dsRNA

and generates more siRNA.

More RdRP is activated and

more dsRNA is made.

RISC complex:RNA-Inducible

Silencing Complex with ATP

hydrolysis.

(RdRP)

(endogenous)


Gene regulation by small RNAs

Dicer gene in C. elegans

siRNAs degrade mRNA

to stop gene expression

quickly

Small temporal (St) RNAs

prevent translation to

stop gene expression quickly


-your RNAi?

--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.


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”


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-mediatedhttp://www.nature.com/nrg/journal/v2/n2/animation/nrg0201_110a_swf_MEDIA1.html

Recruitment of the RING Domain of Cbl to the Epidermal

Growth Factor Receptor (EGFR) Is Essential and Sufficient to

Support Receptor Endocytosis

Fangtian Huang and Alexander Sorkin


Knockdown of growth factor receptor binding protein 2 (Grb2)

by RNA interference strongly inhibits clathrin-mediated

endocytosis of the epidermal growth factor receptor (EGFR).

To gain insights into the function of Grb2 in EGFR

endocytosis, 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 of

Grb2 knockdown on EGFR endocytosis and, moreover,

trafficked together with EGFR during endocytosis.


To generate HeLa cells stably expressing Grb2-YFP, endogenous

Grb2 was knocked down using vector-based short

hairpin RNA (shRNA) with simultaneous expression of

Grb2-YFP that has silencing mutations rendering this construct

insensitive to shRNA.


pSilencer1.0-U6 vector-- endogenous

pSuper-H1 vector--

retrovirus//inducible

Type III RNA pol III promoter

----U6 small nuclear promoter (U6)

----human H1 promoter (H1)


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