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.
2-RNA interference and micro RNA,
4-Transposons and Retroposons,
5-Promoters and Enhancers,
7-RNA Splicing and Processing,
9-Controlling Chromatin Remodeling and Structure.
10-Gene Regulation I,
11-Gene Regulation II,
Molecular Biology of the Cell
Regulatory RNA (review and paper)
Transposons, Retroposons and Retroviruses
Promoters and Enhancers
RNA Splicing and Processing
Controlling Chromatin Structure and Chromatin remodeling
1- Exam= 50% (Parts-I and II)
(review and specific paper, idea, concept and experimental-selected by
(*)-Each student will make a presentation for 15 min. (for specific papers) and 20 min (for review papers) with discussion (2-3 questions).
(Topics plus selected papers)
C. elegans: regulator gene lin4 and its target gene lin14
(lin: Proteins for larval development)
Shooting down mRNA
van der Krol 1990
Gene injection (pigmentation
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
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
--A cellular mechanism that degrades unwanted RNAs in the cytoplasm but not in the nucleus. Why?
--A way for the cell to defend itself.
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)
dsRNA is processed into shorter units (siRNAs) that guide the targeted cleavage of homologous RNA.
--A type of gene regulation
--Involve small RNA molecules
--Induce a double stranded RNA
with RISC (RNA-
the antisense siRNAs act as guides for RISC to associate with complimentary single-stranded mRNAs.Step 3
RISC cuts the mRNA approximately in the middle of the region paired with the siRNA
The mRNA is degraded furtherStep 4
Catalysis: RdRP copies
RNA making more ds RNA.
Dicer complex: RNAase III
with ATP hydrolysis
Dicer cuts, unwinds dsRNA
and generates more siRNA.
More RdRP is activated and
more dsRNA is made.
Silencing Complex with ATP
Dicer gene in C. elegans
siRNAs degrade mRNA
to stop gene expression
Small temporal (St) RNAs
prevent translation to
stop gene expression quickly
--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.
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
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
In these cells, Grb2-YFP fully reversed the inhibitory effect of
Grb2 knockdown on EGFR endocytosis and, moreover,
trafficked together with EGFR during endocytosis.
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
Type III RNA pol III promoter
----U6 small nuclear promoter (U6)
----human H1 promoter (H1)