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miRNA Appear in non-coding regions Primary miRNAs are transcribed by RPII or RPIII Drosha cleaves off cap and poly-A tail to form pre-miRNA After export to cytoplasm, Dicer cleaves stem-loop to form mature miRNA duplex Dicer also ‘selects’ the guide strand for incorporation into the RISC complex -microRNAs are short segments of RNA, 22-25 nucleotides long and initially they’re coded for just within the genome, and they’re found in non-coding regions, specifically, so they can be found in -intron regions of protein coding genes or exon regions of non-protein coding genes -The first step to producing microRNAs is just for them to be transcribed by RP RP II in most eukaryotes, RPIII in some viruses -Upon transcription, the mRNA takes on a secondary structure. It forms a stem-loop precursor with a complementary region which is referred to as the primary microRNA. At this stage, the pri-miRNA has a cap and a poly-A tail. -The primary-microRNA is recognized by the Rnase Drosha, which recognizes the stem-loop, and cleaves off the cap and poly A tail, leaving a stem loop intermediate referred to as the pre-miRNA. The pre-miRNA has a 3’ overhang which comes into play a bit later. -In the meantime, the pre-miRNA gets exported from the nucleus, and then in the cytoplasm, the Rnase Dicer recognizes the 3’ overhang and cleaves it further, taking the stem loop off, leaving a mature microRNA duplex. -Dicer is also involved with selecting one of the strands, the one with lower 5’ thermodynamic stability, to be the guide strand in the RISC complex -RISC = RNA induced silencing complex http://en.wikipedia.org/wiki/MicroRNA
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microRNA mediated host-virus interactions
What Im presenting today is the highlights of a literature review
that Im doing for this class on microRNAs and the role that
microRNA-mediated pathways play in host-virus
interactions.Essentially Ive been reading up on different ways that
microRNAs are used by both the host and the virus to exercise some
control over gene expression, and Ive also been surveying what kind
of experiments were conducted to shed light on these roles of
microRNAs in infected cells. miRNA Appear in non-coding
regions
Primary miRNAs are transcribed by RPII or RPIII Drosha cleaves off
cap and poly-A tail to form pre-miRNA After export to cytoplasm,
Dicer cleaves stem-loop to form mature miRNA duplex Dicer also
selects the guide strand for incorporation into the RISC complex
-microRNAs are short segments of RNA, nucleotides long and
initially theyre coded for just within the genome, and theyre found
in non-coding regions, specifically, so they can be found in
-intron regions of protein coding genes or exon regions of
non-protein coding genes -The first step to producing microRNAs is
just for them to be transcribed by RP RP II in most eukaryotes,
RPIII in some viruses -Upon transcription, the mRNA takes on a
secondary structure. It forms a stem-loop precursor with a
complementary region which is referred to as the primary
microRNA.At this stage, the pri-miRNA has a cap and a poly-A tail.
-The primary-microRNA is recognized by the Rnase Drosha, which
recognizes the stem-loop, and cleaves off the cap and poly A tail,
leaving a stem loop intermediate referred to as the pre-miRNA.The
pre-miRNA has a 3 overhang which comes into play a bit later. -In
the meantime, the pre-miRNA gets exported from the nucleus, and
then in the cytoplasm, the Rnase Dicer recognizes the 3 overhang
and cleaves it further, taking the stem loop off, leaving a mature
microRNA duplex. -Dicer is also involved with selecting one of the
strands, the one with lower 5 thermodynamic stability, to be the
guide strand in the RISC complex -RISC = RNA induced silencing
complex PTGS / RNAi binds 3 UTR translational repression or target
degradation -Now once the RISC complex is formed, youre getting
into whats called the PTGS, or Post transcriptional Gene Silencing
pathway in plants or the RNAi or RNA interference pathway in
animals. -In the RISC complex, the microRNA acts as a probe to
guide the complex to its target.It binds in the 3 un-translated
region and causes either translational repression or mRNA
degradation of the target. -The outcome of the binding is, in fact,
dictated by the degree of complementarity between the miRNA and the
target; perfect complementarity results in target degradation
whereas imperfect complementarity results in translational
repression. -And thats your crash course on miRNA.After that its
just a matter of taking their function and mechanism of action and
putting them in the context of an infected cell and sort piecing
together how that function comes into play in terms of the virus
life cycle and the host cells survival Roles of miRNA -When you do
so, things can get a little complicated.
-Its been known for a while that cells produce microRNAs and that
these can function as negative feedback inhibitors of gene
expression just within the cell. -Its also been known that,
particularly in plants, since they dont have an immune system per
se, host microRNAs have served as a defense mechanism against
viruses -More recently, however, its been shown that viruses also
encode microRNAs, which can be processed by the viruss own
machinery or by the cells machinery if they can sneak in with the
cells miRNAs for processing. -part of the process of identifying
putative viral miRNAs has been through the use of computer programs
that use structural predictions to identify regions that may
correspond to microRNAs -and then the other part has been taking
these hypothesized miRNAs to the bench and verifying whether or not
they actually exist and what they act on. Roles of miRNA Examples
Host miRNA Viral miRNA
In an infected cell youll have Host miRNA Regulate host gene
expression Antiviral defense and is looking to suppress viral gene
expression Viral miRNA Regulate viral gene expression Regulate host
gene expression by suppressing host defense mechanisms Add into the
mix that some viruses have been found to produce proteins that act
as counterdefense by suppressing the RNAi pathwayso theyre
effectively suppressing the suppressors SO you end up with a very
dynamic system and the outcome at any given time is a net effect of
these opposing effectors. Roles of miRNA Host miRNA Regulating host
gene expression Antiviral defense Viral miRNA Regulating viral gene
expression Targets for silencing suppressors Examples 5 HIV-1
HIV-encoded miRNA suppresses transcription of HIVs nef gene ,
acting as a negative regulator of transcription the microRNA is
acting as a negative regulator of transcription -This was
investigated by taking a virus that doesnt have the nef gene,
inserting a plasmid with nef in it, and then coupling the
transcription of the nef gene to the production of beta gal using a
reporter plasmid. -The results here are showing the relative
activity of the reporter gene when the cell is transduced with a
plasmids containing either the specific miRNA, or some other gene.
-What they found was that the samples transduced with the miRNA had
the lowest relative activity, showing that a) that miRNA is
suppressing the gene and b) its doing so at the transcriptional
level. 6 HIV-1 HIV-encoded miRNA suppresses transcription of HIVs
nef gene , acting as a negative regulator of transcription the
microRNA is acting as a negative regulator of transcription -This
was investigated by taking a virus that doesnt have the nef gene,
inserting a plasmid with nef in it, and then coupling the
transcription of the nef gene to the production of beta gal using a
reporter plasmid. -The results here are showing the relative
activity of the reporter gene when the cell is transduced with a
plasmids containing either the specific miRNA, or some other gene.
-What they found was that the samples transduced with the miRNA had
the lowest relative activity, showing that a) that miRNA is
suppressing the gene and b) its doing so at the transcriptional
level. 7 EBV EBV-encoded miRNAs suppress expression of an
interferon-inducible cellular protein that attracts T-cells -An
instance of miRNAs acting as immunosuppressants by downregulating
expression of a host gene -Just some quick background info on EBV,
infection by EBV results in viral latency.And viral latency can be
one of a few different types, and these different types of latency
are defined by the gene expression patterns they exhibit. -HOW: So
these researchers first showed that one of the EBV-expressed miRNAs
in particular was present at elevated levels in cells lines that
had one of the latent forms of the virus.This was quantified by a
Rnase protection assay using P32 labeled RNA probes made from the
target sequence. -HOW: they then investigated the effect of this
miRNA on its target protein by taking latent cells known to express
high amounts of that particular miRNA and transfecting them with
increasing amounts of an oligonucleotide antisense to miRNA, and
this was found to induce expression of the target gene. PICTURE
figure 4B lanes 6-9 Silencing Suppressors
A number of plant viruses produce proteins that target various
steps in the miRNA-induced RNA silencing pathway A number of plant
viruses produce proteins that target various steps in the
miRNA-induced RNA silencing pathway Host defense (Ryu) Silencing
Suppressors
A number of plant viruses produce proteins that target various
steps in the miRNA-induced RNA silencing pathway -HOW: this was
established by inserting ORFs for the suppressor proteins into a
system in which a gene called Chalcone Synthase is constitutively
silenced by miRNA. -The chalcone synthase gene encodes a protein
involved in the production of purple dyes, making the activity of
the gene indirectly measurable, and they also did RNA gel blot
analysis. In this artificial system, it was shown that these
proteins did alter the expression of the Chalcone Synthase gene.
Ruh roh! 10 References Dunoyer, Patrice, Charles-Henri Lecellier,
Eneida Abreu Parizotto, Christophe Himber, and Olivier Voinnet.
"Probing the MicroRna and Small Interfering RNA Pathways with
Virus-encoded Suppressors of RNA Silencing." The Plant Cell.
16(2004): Omoto, Shinya, and Yoichi Fujii. "Regulation of human
immunodeficiency virus 1." Journal of General Virology 86(2005):
Provost, Patrick, Corinna Barat, Isabelle Plante, and Michel J.
Tremblay. "HIV-1 and the microRNA-guided silencing pathway: An
intricate and multifaceted encounter." Virus Research. 121(2006):
Scaria, Vinod, Manoj Hariharan, Souvik Maiti, Beena Pillai and
Samir K. Brahmachari. "Host-virus interaction: a new role for
microRNAs." Retrovirology. 3(2006): Triboulet, Robinson, Bernard
Mari, Yea-Lih Lin, Christine Chable-Bessia, Yamina Benasser, Kevin
Lebrigand, et.al. "Suppresion of MicroRNA-Silencing Pathway by
HIV-1 During Virus Replication." Science. 315(2007): Xia, Tianli,
Andrea O'Hara, Iguaracyra Araujo, Jose Barreto, Eny Carvalho and
Jose Bahia Sapucaia. "EBV MicroRNAs in Primary Lymphomas and
Targeting of CXCL-11 by ebv-mir-BHRF1-3." Cancer Research.
68(2008):