BIL 250 Chapter 17 review Part 2
Teh LV regions are located about
6 kilobases away from thE joining region, a region that contains five possible J encoding exons
Although researchers examined only 1 percent of the genome, they identified more than
1000 direct physical interactions between promoters and distal regulatory elements such as enhancers in each cell type explained Approimatley 60 percent of these interactions were specific to only one cell type
The drosophila genome contains about
13000 gens but dscam gene can produce 2.5 times that many proteins
The binding sites include approximately
40000 enhancers elements and 100000 promoters
each of the light and heavy chains contains
a constant region at the C terminus and a variable region at the N terminus
The next gene in the cascade, doublesex is
a critical control point in the development of sexual phenotype It produces a functional mrNA and protein in both females and males However, the pre mRNA is processed in a sex psecific matter to produce differnt trasnciprts and hence differnt DSX protiesn
In this case,the term gene would encompass
a higher order concept that includes all the senses coding RNAS that contribute to a specific phenotype
The presence of active p53 triggers
a negative feedback loop that creates more mdm2 protein, which rapidly returns p53 to its rare and inactive state
ubiquitin
a small protein that tags other proteins for degradation by proteolytic enzymes.
In females, the functional TRA protein acts as
a splicing factor that binds to the dsx pre mRNA and directs splicing in a female specific pattern
ARE
a stretch of nucleotides that consist of A and U ribonucleotides These AU rich elements are usually located in the 3' untranslated regions of mrNAs that have short, regulated half lives These ARE containing mRNAs encode proteins that are involved in cell growth or transcription control and need to be rapidly modulated in abundance
The sum of the sxl gene acts as
a switch that selects the pathway of sexual development by controlling splicing of the dsx transcript
scientists have discovered that
abnormal activities of miRNAs can tigbe to the occurrence of cancers, diabetes and heart disease
Alternative splicing is found in
all metazoans but is especially common in vertebrates, including humans
The researchers speculate that
all of these multiple interactions between enhancer snp rotors likely do not occur simultaneously within the same cell but reflex the sum of interactions that are occurring within a population of cells that as assaye dodge in individual experiments
short RNAs act in the nucleus to
alter chromatin structure and bring about repression of transcription
The abundance of elements as well as the discovery of the extent of therin eitneraitons promises to
alter our definition of a gene and our under naidn of how the information in DNA is transformed into phenotype
Only about 7 percent of looping interactions occur between
an enhancer and the enares promoter
Adjacent to the J region is
an enhancer seunde that stimulates transcription from any primer located nearby and a single C exon that encodes the constant region of the k light chain molecule
In unstressed cells, p53 is bound by
another protein called Mdm2
It is estimated that
approximately 10 percent of mammalian mrnas contain the instability elements
This is in contrast to the
approximately 20000 protein coding genes that occupy less than 2 percent of the genome
Within the 125 cell types explained
approximately 30 percent of these DNA sites are bound by transcription factors in any one cell type Only 3700 DNA sites are found by transcription factors in all cell types
Approximately two thirds of protein coding genes express
at least two different splicing variants, with an average of four alternative transcripts per locus
Scientists estimate that
at least two thirds of protein coding genes in the human genome can undergo alternative splicing Thus, humans can produce several hundred thousand different points from the appxomiatley 20,000 genes in the haploid genome
75 percent of the genome can
be transcribed
Mdm2 protein
binds to the transcriptional activation domain of the p53 molecule, blocking its ability to induce transitions In addition, Mdm2 acts as a ubiquitin ligase, adding ubiquitin residues onto the p53 protein
The female DSX protein and the male portion are
both transcription factors, but u act in different ways
The presence in females or the absence in males of xl proteins begins
cascades of pre mRNA splicing events that are specific for females or males
DM2
caused by an expansion of the repeat sequence CCTG within the first intron of the ZNF9 gene Affected individuals may have up to 11,000 copies of the repeat sequence in the ZNF9 intron In DM2, the severity of symptoms is not related to the number of repeats
DM1
caused by the expansion of the trinucleotide repeat CTG in the 3' untranslated region of the DMPK gene In unaffected individuals, the DMPK gene contains between 5 and 35 copies of the CTG repeat sequence whereas in DM1 patients the gene contains between 150 and 2000 copies The severity of the symptoms is directly related to the number of copies of each repeat sequence
Overall, the vast majority of transposition in a cell do not
code for points and remain within the nucleus
Each of these steps can be regulated to
control the quantity of a protein product In addition, the stability, modifications and activities of the protein product can be regulated
Treatment of these tumors with synthetic mirANs may be able to
correct these defects and reeves cancer phenotypes
antigens
defined as molecules, usually proteins, which bring about an immune response
All mRNA molecules are
degraded at some point after their synthesis, but the lifetime of an mrNA, defined in terms of its half life, can vary widely between different mRNAs and can be regulated in response to the needs of the cell
siRNAs
derived from longer RNA molecules that are linear, double stranded and located in the cell cytoplasm In nature, these siNRA precursors arise within cells as a result of virus infection or the expression of transposons - both of which synthesize double stranded rNA molecules as part of their life cycles
miRNAs
derived from single stranded rNAS that are transcribed within the nucleus from the cells' own neon and that contain a double stranded stem loop structure Nuclease enzymes within the nucleus recognize these stem loop structures and cleave them from the longer single stranded rNA The stem loop RNA fragments are exported from the nucleus into the cytoplasm where they are further processed by the dicer complex into shorts linear, double stranded miRNAS
Alternative splicing can generate
different forms of mRNA from identical pre mRNA molecules, sot hat expression of one gene can give rise to a number of proteins with similar or different functions
So far, more than 1500 mirNAS have been
discovered in the human genome This is probably an underestimate and scientists speculate that eukaryotic genomes may contain thousands of genes that are transcribed into short noncoding rNAS which may regulate the expression of more than half of all protein coding genes
Scientists have shown that
each neuron expresses a different subset of dscam protein isoforms
In addition, RNA molecules can be
edited and bases can be modified
In 2003, the US national human genome research institute launched the
encyclopedia of DNA elements or ENCODE project
ENCODE data show that
enhancers can influence the transcription of several different promoters and not necessarily the closest one
Hyper Mutation introduces
even more vaition into the LV Legion's sequence
Teh mature messenger RNA is then
exported to the cytoplasm, where it is translated and degraded
Splicing variants expressed by each gene are
expressed at different levels with one variant being the predominant one
Amplification of specific genes, such as rRNA genes in xenopus oocytes, allows
expression of these geese at times during development when high levels of these gene products are required
Recent studies show that the
expression profiles the miRNA genes are characteristic of each tumor type This observation may lead to more precise method so diagnosing tumors, predicting their course and planning treatments
Under normal conditions, the levels of p53 are
extremely low in cells and p53 that is present is inactive
In the presence of sxl
female splicing patterns are expressed which override the default male splicing patterns
When the ratio is 1.0
females are reduced
The four viable regions, when combined together in toe the immunoglobulin molecule
form a unique structure that recognizes one specific antigen
these data likely suggest that
genes and their regulatory elements likely interactions. complex and fluid networks
Except for the occurrence of sequence alterations due to mutations
genomic DNA in most organisms remains remarkably stable However, despite this genomic stability, some significant empanel of gene regulation by DNA rearrangement exist
The discovery of small RNA molecules that control gene expression has
given rise to a new fold of research
clinical trials
have also been started to test RNAi in the rtermane of hepatitis B and respiratory syncytial virus infections Other clinical trials are planned for siRNA treatment of influenza and hepatitis C virus infections
In addition, V regions are susceptible to
high rate so frandom somatic muatiotn - hypthermauttion - during B cell deeloment
Mammals are capable of producing
hundreds of millions of different types of antibodies in response to the presence of a wide variety of angels - more types of antibodies than their agnes in techgnome
Two of these polypeptides are
identical light chains and two are identical heavy chains Together, they form a Y shaped immunoglobulin structure
A total of 128000 transcripts were
identified and more than 70000 for these arise either from DNA in intergenic region sor from antisense transcription within protein coding genes More than 7000 small rNAS were identified including small nuclear RNAS, small nucleolar RNAS, miro rNAS, and transfer RNAS
The goal of ENCODE is to
identify all the functional DNA sequences that lie within the 3.1 billion nucleotides of the human genome and to temerin who these elements act to regulate gene expression
Teh SXL protein is produced only
in embryos with an X:A ratio of 1
Alternative splicing increases
increases the number of genes that can be made from each gene As a result, the number of proteins that can organism can make - its roteome - is not the same as the number of genes in the genome and protein diversity can exceed gene number by an order of magnitude
An added level of control is that p53 is a transcription factor that
induces the transcription of the mdm2 gene
The immune system protects organisms against
infection and the presence of forgien substance that may enter blood or body tissue
Fire and Mello experimental protocol
injected roundworm cells with either single stranded or double stranded rNA molecules - both containing sequences complementary to the mRNA of the unc-22 gene
ENCODE has examined the
interactions between enhancers and promoter a
Intermediate ratios produce
intersexes
This massive endeavor which isc opening more than 400 scientists around the world
is spontaneously half completed
As regulatory roles are being discovered for many of the small noncoding RNAS as well as some long noncoding RNAs
it is still possible the function will be discovered for more of these RNAS
Another consequence of the ENCODE data is that
it will be necessary to alter our concept of a gene as a discrete unit of inheritance
The steady state level of an mrNA is
its amount in the cells determined by a combination of the rate at which the gene is transcribed and the rate at which the mrna is degraded
RNAi was first discovered during
laboratory research, in studies of plants and animal gene expression
These data also indicate that
major differences exist between cell types as to who their gnomes are regulated with each cell type using different combinations of regulatory elements and binding proteins in order to determine the cells' gene expression phenotype
When the ratio is 0.5
males are produced even when no y chromosome is present
Several dozen scientific supply companies now
manufactured synthetic siRNA molecules of specific ribonucleotide sequence of ruse in research These molecules can be introduced into cultured cells to knock out specific gene products
Changes in splicing patterns can have
many different effects on the translated protein Even small changes can alter the protein's enzymatic activity, receptor binding capacity or protein localization in the cell
Another observation is that genes express
many placing variants simultaneously
Regulation of gene expression occurs at
many points along the pathway from DNA to protein
RNAi may be the
method by which cells recognize these double stranded rNAS and inactive them, protecting the organism from external to internal assaults
In addition, certain cancers appear to have defects in
miNRA gene expression
One of the most surprising discoveries of ENCODE research is that
more than 80 percent of the human genome contains some type of regulatory element or participates in active transcription
Many of these small RNAS originate within
mrAS and other long nocing rnAS
DNA encoding the immunoglobulin gene is not fixed but undergoes
multiple programmed changes, includign deletionsm trnaslaitocatiosn nd random mtuaitons
Besides gene amplifications, several other types of programmed DNA rearrangements
occur during developmental regulation for some eukaryotic genes
There are 70 to 100 different L leader and V variable regions about half
of which are functional and can encode the variable V portion of the K light chain molecule
Appromately 50 percent of promoters physically interact with more than
one distal regulatory element, and some have as many as 20 different interactions Similarly, approximately 10 percent of enhancers interact with more than one promoter an dosm times with as many as ten different promoters
During development of a mature B cell
one of the LV regions along with its promoter is randomly joined by a recombination event to one of the ive J regions This recombination event deletes the entire intervening DNA on chromosome 2
Each B cell synthesizes only
one type of immunoglobulin
in the case of ZNF9
only the CCUG sequence repeat itself accumulates in the nucleus, as the remainder of the intron is degraded after splicing of a gene It appears that the accumulated RNAs bind to and sequester proteins that would normally be involved in regulating the alternative splicing patterns of a large number of other RNAs These rNAs include those whose products are required for the proper functioning of muscle and neural tissue
Scientists have discovered
over 20 genes that are inappropriately spliced in dM1 muscle, ehrat and brain
In theory, any disease caused by
over pexion of a specific gene or even normal expression of an abnormal gene product could be attacked by therapeutic rNAi
Finally,some cancers are characterized by
overexpression or abnormal expression of one or several key proto oncogenes
The presence of ubiquitin on p53 results in
p53 degradation
A more extreme example is provided by
para, another gene expressed in the nervous system of drosophila,
Antibody diversity results in
part from the random recombination often do 35 of 50 different functional LV regions with anyone for five different J regions
In addition, rearrangements that lead to loss of some DNA sequences occur only in
particular types of somatic cells in higher eukaryotes Hence these changes are not passed on to progeny
Although transcriptional control exerts the primary effect on gene regulation in eukaryotes
posttranscriptional regulation plays an equal if not more significant role
In addition to its use in laboratory research, RNAi is being developed as
potential pharmaceutical agent
The recombination event that joins tan LV region to a J region is not
precise and can occur anywhere with a region containing several base pairs
In males, no TRA protein is
present and splicing of the dsx pre mRNA results in a male specific mRNA and protein
Introns were seen to be spliced in
primary transcripts that were still itn process of being transcribed
Prior to translation, eukaryotic mRNA transcripts can be
processed by the removal of non coding introns, the precise splicing together of the remaining exons, eh addition of a cap at the mrNA 5' end and the synthesis of a poly A tail at its 3' end
In these cases, these DNA rearrangements are
programmed and essential for normal expression of the genes involved
Recently, scientists have discovered that DM1 and DM2 are caused not by changes in the protein products of the DMPK or ZNF9 genes, but by
the toxic effects of their repeat containing RNAs
ENCODE scientists propose that
the transcript be considered the bsid unity inheritance, rather than the DNA from which it is transcribed
If RNAi method can target these specific gene products
they might help us treat cancers that have become resistant to other methods such as radiation or chemotherapy
if sxl is absent
tra pre mRNAs are spliced into such a way that a translation stop codon remains in the mature mRNA Translation of this mRNA results in a truncated nonfunctional protein
When sxl protein is present,
tra pre mRNAs are spliced out prepuce mature mNRAs that are translated into functional TRA protein
each LV region is preceded by
transcription initiation sequences
Through alternative splicing
two peptide hormones with different structures, locations and functions are synthesized from the same gene
Because enhancer interactions often skip the nearest gene promoter
unknown mechanism must exist to allow regulatory elements to target the appropriate gene at the right time and places to allow appropriate gene expression
Although the immune system has many parts
we will focus on humoral immunity which involves the production of proteins called immunoglobulins or antibodies that bind directly to antigens
Transcription factors bound to enhancer and silencer elements are thought to
work by interacting with other transcription proteins located at promoters These interactions result in the formation of DNA loop supt o 1 million base pairs in length
Fire and Mello results
Although they expected that the single stranded antisense RNA molecules would suppress unc-22 gene expression b y being to the endogenous sense MNRA, they were surprised to discover that one injection for double stranded unc-22 RNA was 10 to 100 fold more powerful in repressing expression of the unc22 mRNA They studied the phenomenon further and published their results in Nature They reported that the presence of double stranded rNA acts to degrade the mrnA if the mNRA is scompletelnary in sequence to one strnad of the double stranded RNA Only a few molecules of double stranded RNA are needed to bring about the degradation of large amounts of mRNA
similarities between siRNA and miRNA
Both types of RNA are short double stranded molecules between 20 and 25 ribonucleotides long
pathways an mRNA can be degraded Third, an mRNA may be cleaved internally by an endonuclease, having unprotected ends at which encon claes degradation may proceed
Examples of endonucleolytic cleavages are those that occur during nonsense mediated decay and those triggered by RNA interference Nonsense mediated mRNA decay occurs when translation terminates at premature stop codons Endonucleases attack the mRNA near the stop codon leaving unprotected ends that are then degraded by exonuclease Nonsense mediated decay is an important mechanism for removing mutated mRNA molecules that could, if efficiently translated, result in accumulation of aberrant protein products
pathways in which mRNA can be degraded
First, an mRNA may be targeted for degradation by enzymes that shorten the length of the poly A tail Second, decapping enzymes can remove the 7 methylguanosine cap, which also renders the mRNA unstable Third, an mRNA may be cleaved internally by an endonuclease, having unprotected ends at which encon claes degradation may proceed
steps of the RNAi pathway commonalities
First, siRNA or miRNA molecules associate with an enzyme complex called the rNA induced silencing complex or RISC Second, within the RISC, the shorts double stranded rNA s denature and the sense strand is degrade Third, the rNA/RISC complex becomes a functional and highly specific agent of rNAi, seeking out mRNA molecules that are complementary to the antisense rNA contained in the RISC At this point, RNAi can take one of two pathways
RISC pathway
If the antisense rNA in the RISC is perfectly complementary to the mRNA, the RISC will cleave the mRNA The cleaved mRNA within the RISC is not exactly complementary to the mRNA, the RISC complex stays bound to the mRNA, interfering with the ability of ribosomes to translate the mRNA RNAi can silence gene expression by affecting either mrNA stability or translation
RITS pathway
In addition to repressing mRNA translation and triggering mRNA degradation, siRANs and mirNAS can also repress the transcription of specific genes and larger regions of the genome They do this by associating with a different complex - the RNA induced transcriptional silencing complex RITS The antisense RNA strand within the RITZ targets the RITS complex to specific gene promoters or larger regions of chaotic RITS then recruits chromatin modifying enzymes of these regions These enzyme methylates histones and DNA, resulting in heterochromatin formation and subsequent transcriptional silencing As a result of their effects on chromatin mediated gene silencing, miRNA molecules are thought to be involved in epigenetic phenomena such as gene imprinting and X chromosome inactivation
sxl in females and males
In females, transcription factor encoded by genes on the X chromosome are thought to activate transcription of the scl gene In males, the lower concentration of these transcription factors is not sufficient to activate transcription of sxl As a result of this differentiation regulation, the sxl protein is expressed only in female embryos
pathways an mRNA can be degraded First, an mRNA may be targeted for degradation by enzymes that shorten the length of the poly A tail
In newly synthesized mRNAs, the poly A tail is about 200 nucleotides long and binds a protein known as poly A binding protein The binding of the protein to the poly A tail helps to stabilize the mRNA If the poly A tail is shortened to less than 30 nucleotides, the mRNA becomes unstable and acts as a substrate for exonucleases that degrade the RNA in a 3' to 5' or 5' to 3' direction
There are many ways by which the quantity and activity of a gene product can be regulated
In some cases, the translation of an mRNA can be regulated to produce the correct quantity of protein product In other cases, the stability of a protein can be modulated or the protein can be modified after translated to change its structure and affect its activity
splicing of the CT/CGRP primary transcript thyroid cells
In thyroid cells, the CT/CGRP primary transcript is spliced in such a way that the mature mRNA contains the first four exons only In these cells, the exon 4 polyadenylation signal is used to process the mRNA and add the poly A tail This mRNA is translated into the calcitonin peptide, a 32 amino acid peptide hormone that is involved in regulating calcium
Recent studies are demonstrating that
RNA induced gene silencing mechanisms operate during normal development and control the expression of baterins of ngos involved in tissue specific cellular differentiation
ENCODE experiment demonstrate that
RNA splicing occurs during transcription
In thyroid cells, the CT/CGRP primary transcript is
spliced in such a way that the mature mRNA contains the first four exons only
Such defects in the regulation of RNA splicing are known as
spliceopathies
As a consequence, p53 proteins are
stabilized, the levels of p53 increase and the protein in able to act as a transcription factor
In animal models, siRNA molecules have
successfully treated virus infections, eye diseases, cancers and inflammatory bowel disease
Immunoglobulins are
synthesized by a type of blood cell known as a B lymphocyte or B cell which undergoes development and maturation in the bone marrow immunoglobulin molecules consist of four polypeptide chains, held together by disulfide bonds
Viral infections are an obvious
target and science have had promising results using RNAi in tissue cultures to reduce the severity of infection by several types of viruses such as HIV, influenza and polio
Sex in drosphila is determined by
teh ratio fo x chrosomes to sets of autosomes X:A
One well studied mRNA stability element is
that adenine uracil rich element ARE - a stretch of nucleotides that consist of A and U ribonucleotides
in vitro studies have shown
that each dscam protein isoform rna bind to the same dscam protein isoform but not to others Even a small change in amino acid sequence reduces or eliminates the binding between two dscam molecules
An important example of posttranslational regulation is
that of the p53 protein
splicing of the CT/CGRP primary transcript brain and peripheral nervous system
the CT/CGRP primary transcript is spliced to include exons 5 and 6 but not exon 4 In these cells, the exon 6 polyadenylation site is recognized The CGRP mRNA encodes a 37 amino acid peptide with hormonal activity in a wide range of tissues
After this somatic recombination event
the LV promoter is activated by the nearby presence of the immunoglobulin enhancer result gin transcription of a gene The resulting rNA is spliced to remove the introns between the L and V regions and between the J and C regions After translocation of the mature mRNA, the [polypeptide is processed by removal of the ladder manio acis The rearranged K gene is then matintaine and passed on to progeny of that B cell
The steady state level determines
the amount of mrNA that is available for translation
The changes in the levels and activity of p53 are due to
the combination of increased protein stability and modifications to the protein
Because alternative splicing is far more common in vertebrates
the combinations of protein that can be produced from human genome may be astronomical
The regulatory gene at teh beginning of thsi cascade is
the gene sex lethal, which codes an RNA binding protein
The mechanisms of DNA rearaurants described here
the human K light chain genes also occur during the formation of the gene that encodes another type of light chain gene as well as the gen eth encodes the immunoglobulin heavy chains
When cells suffer DNA damage or metabolic stress
the level of p53 protein increase dramatically in addition, p53 becomes an active transcription factor
proteome
the number of proteins that can organism can make
These data suggest
the potential for large amounts of antisense and overlapping transcription initiation through protein coding and noncoding genes
Scientists are particularly enthusiastic about
the potential uses of RNAi in the diagnosis and treatment of cancers
One of the targets of the sxl protein is
the pre mRNA encoded by the transformer gene
The ultimate end point of gene expression is
the presence of an active gene product
Because of this imprecision
the recombination between any particular pair of LV and J regions still shows considerable variation
Two major types of short RNA molecules are involved in RNA induced gene silencing
the smallinterfing rNAs or siRNAS and the micrRNAs or miRNAS
SXL alters
the splicing pattern of tra pre mRNA by binding to the pre mRNA and altering recognition of splicing signals by the splicing machinery
In these studies
the synthetic siRNA molecules were applied to the surfaces of mucous membrane or into eyes, brian tissues or the lower intestine
The human k light gne is assembled during
B cell development from multiple dNA regions located along chromosome 2
Often the fetal splicing patterns occur in
DM1 and DM2 patients by the normal transactions ot adult spelling patterns are lacking
DSX F vs DSX M
DSX F represses the transcipton fo genes whose proudcts contorl male sex deelopment, whereas DSX M activates teh tranciton fp gens whose proudcts contorl male sexual development In addition, DSX M represses the transcription of genes that control female gsexual development
Given the existence of alternative splicing, how many different polypeptides can be derived from the same pre mRNA? Research on the dscam gene in drosophila melanogaster
During development, cells of the nervous system most accurately connect with one another Even in drosophila, with only about 250000 neurons, this is a formidable task Neurons have cellular processes called axons that form connections with other nerve cells The drosophila dscam gene encodes a protein that guides axon growth, ensign that neurons are correctly wired together In dscam pre MRNA, exons 4,69 and 17 eac consist of an array of possible alternatives These are spliced into the mature mRNA in an exclusive fashion, so that each exon is represented by no more than one of its possible alternatives There are 12 alternatives to exon 4, 48 for exon 6, 33 for exon 9 and 2 for exon 17 The number of possible combinations could be forme suggests that theoretically dscam gene can produce 38016 proteins
Within the 80 percent of the genome once considered "junk DNA"
ENCODE has created thousands of enhancers promoters, sequences encoding RNA transcripts and sites of specific chromatin modifications
By examining chromatin features such as DNase hypersensitivity and histone modifications that are charceirisc of DNA involved in regulatory factor binding
ENCODE researchers discovered more than 1 million bidding sites for gene regulation proteins, including transcription factors that bind to enhancers and promoters
enhancer RNA
Enhancer elements often bond tain bonda finde rotmer elements that initiate transcription from the enhancer element and extend outward for several kilobases The most active enhancers in a cell type produces the most enhancer RNA
When cells are stressed
Mdm2 and p53 are modified by phosphorylation and acetylation, resulting in the release of mdm2 form p53
DNA rearrangements
One type of arrangement results in the creation of new genes from gene fragments located at various positions in the genome Another type of rearrangement result isn't a switch in expression of genes as a result of a reaction or DNA copying event
What are the mechanisms by which mRNA stability can be regulated?
One way that an mRNA's half life can be altered is through specific RNA sequence elements that recruit degrading or stabilizing complexes
para gene
Para not only has at least 6 sites of alternative splicing, leading to 48 possible different mRNA variants, but also undergoes another post translational modification called RNA editign, at 11 positions RNA editing involves base substitutions made after transcription and splicing With both alternative splicing and editing, the para gene can theoretically produced more than 1 million different transcripts
studies phase II and III
Phase II and III efficacy trials have begun to test the use of siRNAS directed again the product of the vascular endothelial growth factor gene, as a drug to treat age related macular degeneration
RNAi mediated gene silencing is relatively
specific and inexpensive and it allows scientists to rapidly analyze gene function
studies phase I
Research into RNAi pharmaceutical has made rapid progress in the phase I toxicity stages of the first human clinical trials have been completed
source of siRNA molecules research lab
Scientists are only able to introduce double stranded RNAs into cells for research or therapeutic purposes In the cytoplasm, double stranded RNA molecules are recognized by an enzyme complex known as Dicer and is cleaved by dicer into SiRNAs
The functional relevance of this enormous amount of trwanciptin throughout the genome is intriguing but onctrovsersal
Some scientists argue that it may be simply "noise" without significance to cellular phenotype If this is the case, substantial regulation of gene expression must occur at post transcriptional levels in eukaryotes
p53 protein
The p53 protein is essential to protect normal cells from the effects of dNA damage and other stresses It is a transcription factor that increases the transcription of a number of genes whose products are involved in cell cycle arrest, DNA repair and programmed cell death
RNAi technology allows investors to
specifically create single gene defect without having to induce inherited gene mutations
DM1 and DM2 repeat containing RNAs
These RNAs accumulate and form inclusions within the nucleus
transformer gene
This pre mRNA is transcribed in both male and female cells
RNAi pathways are also able to repress transcription in indirect ways
Transcription factor mRNS are frequent targets for RNAi induced silencing When the levels of specific transcription factors are reduced in a cell, transcription of genes whose expression depends on these factors i slap repressed
Approximately 30000 of the promoter regions found by examining chromatin features were located within
protein coding genes and 17000 of these were oriented in an antisense direction
myotonic dystrophy
provides an example of how defects in alternative splicing can lead to a wide range of symptoms Myotonic dystrophy is the most common form of adult muscular dystrophy, affecting 1 in 8000 individuals It is an autosomal dominant disorder that occurs in two forms DM1 and DM2 Both of these diseases show a wide range of symptoms, including muscle wasting myotonia (difficulty relaxing muscles), insulin resistance, cataracts testicular atrophy, behavioral and cognitive deficits, cardiac muscle problems and hair follicle tumors
In most somatic cells, as well as in germ cells, these DNA regions are
recognized as shown
It does this by
recognizing molecules on the surface of these forigen substances and by physically bind it to them in a kind of lock and key configuration - a phenomenon referred to as antigen recognition
First discovered in plants, short RNA molecules are known to
regulate gene expression in the cytoplasm of plants, animals and fungi by repressing translation and triggering the degradation of mrNAs This form of sequence specific post transcriptional regulation is known as RNA interference or RNAi
In cells not growing or
require low levels of gene expression, specific complexes bind to the AREAS of these mRNA molecules, bringing about shortening of the poly A tail and rapid mRNA degradation
Failure to control the spacing the dsx transcript in a female model
results in the default splicing of the transcript in a mal model, leading to the production of a male phenotype
These RNAs are transcribed from
sequences within the introns of teacher protein coding genes or form their own promoters located within noncoding regions of the genome
Mutations that affect regulation of splicing contribute to
several genetic disorders
Three major genes in this pathway are
sex lethal, transfer and doublesex
RNA induced gene silencing
short RNAs in the nucleus alter chromatin structure to repress transcription
in vivo studies
show that cells expressing the same isoforms of dscam interact with each other Therefore, it appears that the diversity of dscam protein isoforms in neurons prides a kind of molecular identity tag for each neuron, helping to guide it the correct target and preventing the tagline of extensions from different neurons
Over the last few years, scientists have discovered that
significant amounts of eukaryotic genomes are transcribed by RNAP II into rNA products that contain no open reading frames and are not translated into protein products
Chromosomal ratios are intrepid by a
small number of genes that initiate a cascade of splicing events, resulting in the production of male o female somatic cells and the corresponding male or female phenotypes
