Welcome Each of You to My Molecular Biology Class

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Welcome Each of You to My Molecular Biology Class
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Molecular Biology of the Gene, 5/E --- Watson et
al. (2004)
Part I Chemistry and Genetics Part II
Maintenance of the Genome Part III Expression
of the Genome Part IV Regulation Part V Methods
2005-5-10
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• Chapter 16 Regulation principles and How genes
  are regulated in bacteria
• Chapter 17 Basic mechanism of gene expression in
  eukaryotes
• Chapter 18 The mechanism of RNAi and the role of
  miRNA in development and cancer

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• Molecular Biology Course

• Chapter 8
• RNA in regulation
• The mechanism of RNAi and the role of miRNA in
  development and cancer

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CHAPTER 18 RNAi and miRNA in development and
cancergenesis
??RNA??????
Topic 1 RNA interference and its mechanism
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1 Double-stranded RNA inhibits expression of
genes homologous to that RNA. phenomena-??
??RNA?????????????
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2006????????????
Andrew Z. Fire Craig C. Mello
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Fig 2. Analysis of RNA-interference effects in
individual cells. Fluorescence micrographs show
progeny of injected animals from GFP-reporter
strain PD4251 (a C. elegans strain expressing GFP
fluorescence protein) (???????????).
ds-gfp RNA
Control dsRNA
Young larva (??)
Adult (??)
adult body wall at high magnification
(?????? ????)
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Fig 3. Effects of mex-3 RNA interference on
levels of the endogenous mRNA (in situ
hybridization in embryos) (???????).
hybridization (endogenous mex-3 RNA)
No hybridization and staining
antisense hybridization
ds mex-3 RNA hybridization
adult body wall at high magnification
(?????? ????)
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An plant immune system Virus-induced gene
silencing (???????????). Most plant viruses
have single-stranded RNA genomes, which are
released from the protein coat of their virus
particles as they enter a cell.Their genomic RNA
is then replicated by the virus encoded
RNA-dependent RNA polymerase to produce sense and
antisense RNA, which can hybridize to form dsRNA
and trigger an RNAi response against their own
sequences.
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2. Short interfering RNA (siRNAs) are produced
from dsRNA and direct machinery that switch off
genes in various way. Mechanism-??
???RNA??????RNA??????????????????
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The question to be addressed is Why exogenous
dsRNA can inhibit expression of genes homologous
to that RNA?
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Exogenous dsRNA ????RNA
Figure 17-30 RNAi silencing
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• The targets of the RNAi-directed gene silencing
• Degradation of the target mRNA (????mRNA???),
• Inhibition of translation of the target mRNA
  (????mRNA???),
• Silencing the gene transcription from the target
  promoter (????????????).

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• The heart of the RNAi mechanism
• Dicer an RNaseIII-like multidomain ribonuclease
  that first processes input dsRNA into small
  fragments called short interfering RNAs (siRNAs)
  or microRNAs (miRNA). Dicer then helps load its
  small RNA products into RISC.
• RISC (RNA induced silencing complexes)
  (RNA????????) a large multiprotein complex that
  direct the bound siRNA or miRNA to its target and
  inhibit the target gene expression.

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Dicer
Structural organization ---A PAZ domain, binds
the end of the dsRNA ---Two RNase III
domains ---Other non-conserved domains.
?????
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The crystal structure of the Giardia intact Dicer
enzyme shows that the PAZ domain, a module that
binds the end of dsRNA, is separated from the two
catalytic RNase III domains by a flat, positively
charged surface. The 65 angstrom distance
between the PAZ and RNase III domains matches the
length spanned by 25 base pairs of RNA. Thus,
Dicer itself is a molecular ruler that recognizes
dsRNA and cleaves a specified distance from the
helical end.
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RISC the key component is Argonaute (AGO)
AGO2
TRBP
Dicer
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Argonaute (AGO) A large protein family that
constitutes key components of RISCs. ---AGO
proteins are characterized by two unique domains,
PAZ and PIWI, whose functions are not fully
understood. Current evidence suggests that the
PAZ domain binds the 3-end two-nucleotide
overhangs of the siRNA duplex, whereas the PIWI
domain of some AGO proteins confers slicer
activity. PAZ and PIWI domains are both essential
to guide the interaction between the siRNA and
the target mRNA for cleavage or translational
repression. ---Distinct AGO members have
distinct functions. For example, human AGO2
programs RISCs to cleave the mRNA target, whereas
AGO1 and AGO3 do not.
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A model for siRNA-guidedmRNA cleavage by
Argonaute
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The multiple functions of RNAi
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3. MicroRNA (miRNA) its processing
??RNA????
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MicroRNA (miRNA) A type of non-coding small RNA
(2123 nucleotides) produced by Dicer from a
stem-loop structured RNA precursor (70-90 nts
ong) (?????). miRNAs are widely expressed in
animal and plant cells as RNAprotein complexes,
termed miRISCs, and have been implicated in the
control of development because they lead to the
destruction or translational suppression of
target mRNAs with homology to the miRNA
(????????).
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Structure of pri-miRNAs
Pri-miRNAs bear the 5 cap and 3 poly(A) tails
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miRNA processing
Pri-miRNA (miRNA??????) Drosha (1) pre-miRNA (mi
RNA??) Dicer (2) miRNA
Exportin 5 (Exp5) transports pre-miRNA to the
cytoplasm
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Human Drosha and Dicer share the same RNase III
domains and dsRNA binding domain.
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The number of the identified miRNAs is growing
rapidly in recent years. Over 4000 miRNAs have
been found until the October of this year (The
miRBase Sequence Database). Release 9.0 (Oct,
2006) of the database contains 4361 entries
representing hairpin precursor miRNAs, expressing
4167 mature miRNA products, in primates, rodents,
birds, fish, worms, flies, plants and viruses.
The data are freely available to all through the
web interface at http//microrna.sanger.ac.uk/sequ
ences/ and in flatfile form from
ftp//ftp.sanger.ac.uk/pub/mirbase/sequences/.
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CHAPTER 18 RNAi and miRNA in development and
cancergenesis
??MicroRNA?????????,?????
Topic 2 miRNAs in animal development and other
functions
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1. miRNA in C. elegans development
???? C. elegans
Victor R. Ambros
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lin-4 and let-7 miRNAs control the developmental
time of C. elegans.
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Expression of lin-4 allows C. elegans to proceed
to the late developmental stage
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lin-4 binds its target mRNAs by imperfect base
pairing.
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2. miRNAs in vertebrate development There are a
lot unknown because the the lack of efficient
methods to uncover the targets of miRNAs.
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Learning the miRNA function from its expression
pattern
??
??
??
???
Figure 2. Expression of miR-124a and miR-1 in
Zebrafish, Medaka, Mouse, and Fly. miR-124a is
restrictedly expressed in the brain and the
spinal cord in fish and mouse or to the ventral
nerve cord in the fly. The expression of miR-1 is
restricted to the muscles and the heart in the
mouse.
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miRNA controls some plant phenotype (????????)
Jaw-miRNA ?????????
(Nature, 2003)
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miRNA controls the differentiation of the
hematopoietic stem cell (??????????)
3?miRNA?????????????????
( Science 2004)
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Some viruses encode miRNAs (??????miRNAs)
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CHAPTER 18 RNAi and miRNA in development and
cancergenesis
????RNA?????????
Topic 3 miRNA in cancer
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miRNAs in human There are about 500 miRNAs
from human have been found and annotated. They
are named as has-miRx.
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miRNA expression pattern changes during
oncogenesis, and is unique for each
cancer. ??RNA????????????
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Figure 3, Comparison between normal and tumor
samples reveals global changes in miRNA
expression.
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One mechanism of miRNA controlling oncogene
expression ??RNA?????????????
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• c-Myc is a helixloophelix leucine zipper
  transcription factor that regulates an estimated
  1015 of genes in the human and Drosophila
  genomes.
• c-Myc activates expression of a cluster of six
  miRNAs on human chromosome 13. (Figure 1)
• E2F1 is the transcription factor, which is a
  target of c-Myc that promotes cell cycle
  progression.
• Expression of E2F1 is negatively regulated by two
  miRNAs in this cluster, miR-17-5p and miR-20a.
  (Figure 1)

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Used 2-O-methyl Antisense oligonucleotides to
downregulate the level of miR-17-5p and miR-20a,
and then analyzed the protein (B-Western) and
mRNA levels (C-Northen) of E2F1.
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Some microRNAs are potential oncogenes
????RNA????????
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B-?????
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Figure 1. The mir-1792 cluster shows increased
expression in B-cell lymphoma samples and cell
lines. The level of mir-1792 pri-miRNA was
determined by real-time quantitative RT-PCR in 46
lymphomas and 47 colorectal carcinomas, and
compared to levels found in corresponding normal
tissues from five individuals.
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Figure 2. Overexpression of the mir-1719b
cluster accelerates c-myc-induced lymphomagenesis
in mice.
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CHAPTER 18 RNAi and miRNA in development and
cancergenesis
??siRNA???
Topic 4 siRNA application
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siRNA application in mammalian

• Transfect exogenous siRNA into cells (transient
  expression)
• Chemical synthesis expensive
• In vitro transcription of pre-miRNA with T7
  promoter.
• In vitro transcription of long dsRNA by that are
  then cleaved by E. coli RNase III or RNase
  III-like DICER.
• Expression of siRNA in cultured cells or in
  animal models
• siRNA produced with pol III promoter from the
  transfected DNA plasmids.

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Expression of hairpin RNA (shRNA) using Pol III
promoters

• Transcription from RNAP III promoters of U6 and
  H1 are well characterized. RNAP III transcription
  uses a well-defined termination signal (TTTTT)
  and the products have no extra sequence.
• 2. Transcription from these promoters is very
  efficient in various tissues.

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A mammalian expression vector designed to direct
the intracellular synthesis of siRNAs.
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• Create induced phenotypes that can be observed
  over long time spans
• Create a stably engineered cells can be assayed
  either in vitro or in vivo, perhaps testing the
  angiogenic (????) or metastatic (??) potentials
  of tumor cells in xenograft models (??????)?
• Create hypomorphic alleles (?????) rapidly in
  transgenic mice.

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• 4. Combine shRNAs with existing high-efficiency
  gene delivery vehicles to create bona fide
  RNAi-based therapeutics. For example, ultimately,
  to silence a disease-causing mutant allele
  specifically.

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Research Applications of RNAi A new strategy of
reverse genetics a novel way of gene knock-out

• It can be used in reverse genetics (?????) to
  identify the cellular or biological function of a
  gene.
• It can be combined with genomics to perform
  large-scale genetic screens aimed at gene
  discovery.

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Therapeutic Applications of RNAiA new strategy
to invitation of new drugs and gene therapy

• siRNAs can be used to counter viral infection by
  specifically destroying the mRNAs of the
  pathogenic viruses, such as HIV and HBV.
• siRNAs can be applied to counter cancers by
  specifically down-regulate the expression of
  genes related to oncogenesis.

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Key points of the chapter

• RNA interference siRNA, miRNA and RNAi
  mechanism.
• miRNAs functions animal development etc.
• miRNA in cancer biomarker, mechanisms
• siRNA application methods to produce siRNA, the
  application of siRNA in research and therapeitics.

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???RNA?? ??RNA??,???RNA(siRNA)???RNA(miRNA)???
?RNA????? ???  MicroRNA?????????,????? ????RNA??
?????????? ???  ??RNA????????? ??????RNA?????RN
A????????????????RNA??????????? ???
siRNA??? ??siRNA??????????????????