1. Hours: Tu/Th 2:30 to 4 pm or by appointment.
Molecular
Cellular
Biology-I
(MCB-I)
PCB 6025
M. Alejandro Barbieri

Office: AHC C/214

Hours: Tu/Th 2:30 to 4 pm or by appointment.

2. TOPICS 1-Regulatory RNA /CRISPR-Cas system/RNA interference
and micro RNA,
2-Retroviruses,
3-Transposons and Retroposons,
4-Promoters and Enhancers,
5-Activating Transcription/Epigenetic Effects
6-RNA Splicing and Processing/Enhancer: eRNAs and lncRNAs
7-Chromosomes-Nucleosomes,
8-Controlling Chromatin Remodeling and Structure.
9-Gene Regulation I,
10-Gene Regulation II

3. Books

4. Books
books

5. Presentation: Topic selection/student
Regulatory RNA (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, Prokaryotic Gene Regulation-CRISPR-Cas 9 system
The Mechanisms and Significance of mRNA Degradation
RNA Splicing- SR Proteins

6. Evaluation:
1- Exam= 50%
2-Reading Material= 20 %
3-Presentation =20%
4-Participation = 10 %*
(*)-Each student will make a presentation for min with 5 to 10 min discussion (2-3 questions).

7. Open book “Take home” exam
Close book exam or
Open book “Take home” exam

8. Exam: 50%.
-Exam will be “open” and it will have 25 “essay type” questions and each question will have 3 to 5 sub-questions.
--Each correct answered question is equally weighted (2 points per correct question).
--Exam date and time: It will start at 12:01:00 am on 10/30/17 and it will finish at 11:59:00 pm on 11/5/17.
--No make up Exam will be given, except in cases of unforeseeable and severe documented circumstances beyond your control.

9. Reading Material: 20%.
--Outside of some lectures by the instructors, the course will focus on review articles for discussion (reading material).
--Each student will write a specific “highlights” of the review paper assigned for each lecture.
--The language of each “highlights” should be clear, concise and to the point, and you should use 5 full sentences and each sentence should contain no more then 90 characters.
--Each “highlights” is equally weighted (2.22 points per correct presented “highlights”).
--Highlights deadline: Each highlight has a specific deadline and they can NOT be sent via . Only hard copy will be accepted.
--No make up Highlight will be given, except in cases of unforeseeable and severe documented circumstances beyond your control.

10. Highlights Deadline (12:30 pm)
Genome Engineering 9/5/17
Retrotransposons and genome regulation 9/12/17
Transcription units /19/17
Chromatin and Enhancer 9/26/17
Nuclear organization /3/17
Nucleosome Structure and Function 10/10/17
mRNA degradation and splicing 10/17/17
The type II secretion system 10/24/17
The type III secretion system 10/31/17

11. Presentations: 20% and Participation: 10%.
--Students will choose original research or specific review articles that have been published in 2017 on selected topic assigned by the instructor (see specific topics section in the syllabus section).
--Students must send 5 articles to the instructor, who will select the article to be presented no later than 9/19/17. The Instructor will then send the selected article for each student no later then 9/26/17 together with the exact day of the presentation.

12. --Before each presentation (24 hrs), the presenter has to to the whole class and instructor a ppt/pdf file of the PowerPoint presentation along with research paper. Each student will make a presentation (~25 minutes) on the research articles.
--Preparation for the presentation involves knowing well the article as well as any supplemental material.
---Presentation of the data should involve getting to know how the experiments were done so that data can be critically analyzed.

13. Regulatory RNA

14. 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)

15. RNA Interference Is Related to Gene Silencing
RNA interference triggers degradation of mRNAs complementary to either strand of a short dsRNA.
Figure 13.21

16. dsRNA may cause silencing of host genes: no more proteins
NK,
p38,
NFkB,
PP2A
Stress
Signal
2’, 5’-oligoadenylate synthetase (2’, 5’-AS)

17. What is RNA interference?
Shooting down mRNA

18. RNAi Background What is it? Why use it? The mechanism and process
Experimental considerations

19. Plasmid
Virus

21. Bill Douherty and Lindbo 1993 Hamilton and Baulcombe 1998
Jorgensen 1990
van der Krol 1990
Gene injection (pigmentation
Enzyme-petunias)
Expectation: more red color
Co-suppression of transgene
and endogenous gene.
Bill Douherty and Lindbo 1993
Hamilton and Baulcombe 1998
Gene injection with a complete tobacco
etch virus particle.
Expectation: virus expression
Co-suppression of transgene
and virus particles via RNA.
Identification of short antisense RNA
sequences
dsRNA?
How?
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

22. Shooting mRNA means RNA interference

23. 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.

24. 2. Works in any cell/organism 3. Uses conserved endogenous machinery
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.

25. The mechanism of small interfering RNAs (siRNAs)
What happens?
dsRNA is processed into shorter units (siRNAs) that guide the targeted cleavage of homologous RNA.

26. The RNAi process RNA interference: --A type of gene regulation
--Involve small RNA molecules
--Induce a double stranded RNA
The RNAi process

27. 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)

28. Step 2 The siRNAs associate with RISC (RNA- induced silencing
complex) and
unwind

29. Step 3
the antisense siRNAs act as guides for RISC to associate with complimentary single-stranded mRNAs.

30. Step 4
RISC cuts the mRNA approximately in the middle of the region paired with the siRNA
The mRNA is degraded further

32. SiRNP: small interfering ribonucleoprotein particle
ssRNA (exogenous)
Exam
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)
Exam
(endogenous)
SiRNP: small interfering ribonucleoprotein particle

33. 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

34. -your RNAi?
--MicroRNAs (miRNA) are single-stranded RNA molecules of about 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.

36. Experimental Considerations
Transfection method:
1-Lipofectamine 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”

38. Growth Factor Receptor Binding Protein (Grb) 2-mediated
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

40. Knockdown of Grb-2 by RNAi inhibited internalization of EGFR.
We have generated cell lines in which endogenous Grb2 was
replaced by a modified (YFP)-tagged Grb2
YFP-Grb2 was expressed at the physiological level
Grb2-YFP fully reversed the inhibitory effect of
Grb2 knockdown on EGFR endocytosis

41. 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.

42. retrovirus//inducible
pSilencer1.0-U6 vector--
pSuper-H1 vector--
retrovirus//inducible
Type III RNA pol III promoter
----U6 small nuclear promoter (U6)
----human H1 promoter (H1)

43. Ets2 maintains hTERT gene expression and breast cancer cell proliferation by interacting with c-Myc.
Xu D, Dwyer J, Li H, Duan W, Liu JP.

45. Philadelphia chromosome:
chronic myelogenous leukemia (CML).
ABL: kinase, BCR: Substrate