Dynamics of Genetic Environment and Ecology

[Guest guest]

> Life after the Central Dogma

>

> The biotech industry was launched on the scientific

> myth

> that organisms are hardwired in their genes, a myth

> thoroughly exploded by scientific findings

> accumulating

> since the mid 1970s and especially so since genome

> sequences

> have been accumulating (see Living with the Fluid

> Genome

> http://www.i-sis.org.uk/fluidGenome.php, by Mae-Wan

> Ho ).

>

> We bring you the latest surprises that tell you why

> our

> health and environmental policies based on genetic

> engineering and genomics are completely misguided;

> and more

> importantly, why the new genetics demands a

> thoroughly

> ecological approach.

>

> Death of the Central Dogma

> http://www.i-sis.org.uk/DCD.php

> Caring Mothers Reduce Response to Stress for Life

> http://www.i-sis.org.uk/MCDIRTS.php

> Subverting the Genetic

> Text http://www.i-sis.org.uk/RNASTGT.php

>

>

>

> ISIS Press Release 09/09/04

>

> Subverting the Genetic Text

> *********************

>

> Dr. Mae-Wan Ho exposes the hidden

> intrigues in the vast RNA underworld where layers of

>

> interference and machinations subvert the chain of

> command

> from DNA to RNA to protein.

>

> The references

> http://www.i-sis.org.uk/full/RNASTGTFull.php

> and diagram in Figure 1 is posted on ISIS members'

> website.

> Details here http://www.i-sis.org.uk/membership.php.

>

> Updating and re-interpreting the sacred text

>

> According to the Central Dogma, DNA, the genetic

> text, is

> read out into RNA and RNA is translated into

> protein. RNA is

> rather like the scribe copying and translating the

> sacred

> text to direct the faithful. But geneticists are now

>

> uncovering a vast underworld of heresy to the

> Central Dogma

> where RNA agents not only decide which bits of text

> to copy,

> which copies get destroyed, which bits to delete and

> splice

> together, which copies to be transformed into a

> totally

> different message and finally, which resulting

> message -

> that may bear little resemblance to the original

> text - gets

> translated into protein. RNAs even get to decide

> which parts

> of the sacred text to rewrite or corrupt. The whole

> RNA

> underworld also resembles an enormous espionage

> network in

> which genetic information is stolen, or gets

> re-routed as it

> is transmitted, or transformed, corrupted,

> destroyed, and in

> some cases, returned to the source file in a totally

>

> different form.

>

> And this underworld is big, really big. The

> protein-coding

> sequence is only about 1.5% of the human genome.

> Yet, around

> 97 - 98% of the transcriptional readout of the human

> genome

> is non-protein-coding RNA. This estimate is based on

> the

> fact that intronic RNA makes up 95% of the primary

> protein-

> coding transcripts on average, and there are large

> numbers

> of non-coding RNA transcripts which may represent at

> least

> half of all transcripts. Most of the miRNAs

> (microRNA, see

> below), for example, are derived from (intergenic)

> regions

> between genes; and almost half of all transcripts

> from the

> mouse genome are non-coding RNAs. A similar estimate

> applies

> to the human genome [1].

>

> The inescapable conclusion is that the job of

> mediating

> between DNA and protein is really the centre stage

> of

> molecular life. And who gives orders to the

> multitudes of

> RNA agents? In a sense it is everyone and no one,

> because

> the system works by perfect intercommunication. It

> is not

> the DNA, but rather, the particular environment in

> which the

> RNA agents find themselves. For the organism

> (organization)

> to survive, it needs to turnover the DNA text

> continuously,

> adapting to the realities of its environment. In the

>

> process, it keeps certain texts invariant (see " Are

> ultra-

> conserved elements indispensable? " this series),

> while

> changing others rapidly in non-random ways (see " To

> mutate

> or not to mutate " , this series). It also needs to

> keep

> referring to texts that are relevant, modifying it,

> or

> updating the interpretation in keeping with the

> times (see

> " Keeping in concert " this series).

>

> RNA interference

>

> RNA interference (RNAi) was first discovered in the

> nematode

> worm, C. elegans in the 1990s. Researchers noticed

> that

> injecting either sense RNA (the sequence that gets

> read and

> translated into protein) or antisense RNA (the

> complementary

> sequence, which does not code for protein) into the

> worm led

> to specific silencing of the gene involved. It was

> later

> found that the phenomenon was actually caused by

> double-

> stranded RNA (dsRNA) contaminating the sense or

> antisense

> RNA. RNAi now refers to all gene-silencing induced

> by dsRNA.

>

> These include a host of other phenomena discovered

> at around

> the same time [2, 3]. For example, a gene could be

> silenced,

> or 'co-suppressed', simply by introducing an extra

> copy into

> the genome as a transgene, and transgenes themselves

> may be

> silenced either at or after transcription. The coat

> protein

> gene of a virus transferred into a plant may protect

> the

> plant from the virus, by silencing the virus' genes.

>

>

> All these phenomena are interlinked through special

> pathways

> of RNA processing that are only just being defined

> (see Fig.

> 1). Abnormal single stranded RNA (ssRNA) is turned

> into a

> double stranded RNA (dsRNA) by an RNA-dependent RNA

> polymerase enzyme (RDRP). The dsRNA is then chopped

> up into

> small pieces or microRNA (miRNA) by the enzyme

> Dicer. The

> same enzyme also processes certain hairpin RNA

> (hpRNA) and

> related pre-microRNA (pre-miRNA) into miRNA. The

> miRNA is

> further processed into single-stranded RNA that's

> incorporated into a multiprotein complex called

> RNA-induced

> silencing complex (RISC). At this point, the single

> stranded

> RNA fragment binds to complementary part of the

> messenger

> RNA and either causes the breakdown of the mRNA or

> prevents

> its translation into protein. Remember that all this

> depends

> on complementary base pairing, just as in DNA, so

> these

> mechanisms could potentially exist for each and

> every one of

> the now estimated 24 500 genes in the genome.

>

> Figure 1. RNA interference pathways

>

> It turns out that dsRNA is not only involved in

> signalling

> the breakdown or inactivation of specific mRNA to

> prevent

> the _expression of the protein coded, it is also

> involved in

> triggering anti-viral response in mammals. And this

> is a

> major obstacle to achieving RNAi in mammals, which

> might be

> useful in silencing specific genes in gene therapy.

> Double-

> stranded RNAs longer than 30 nt (nucleotide)

> activate an

> antiviral response that includes the production of

> interferon, resulting in the non-specific breakdown

> of RNA

> transcripts and a general shutdown of protein

> synthesis. In

> order to overcome this obstacle, synthetic 21nt

> miRNAs have

> been used. These are long enough to induce

> gene-specific

> suppression and short enough to evade host

> interferon

> response. However, recent work has shown that under

> certain

> conditions, even such small miRNAs can activate the

> interferon system. One activating signal for the

> interferon

> response appears to be the triphosphate group at the

> 5' end

> of the miRNA synthesized by a phage polymerase [4].

> In

> addition, there are other problems, such as avoiding

>

> interfering with non-target sequences [5],

> especially as

> perfect base-pairing is not required, and matches of

> as few

> as 11 consecutive nucleotides can give non-target

> effects.

>

> RNA-directed DNA read-out

>

> The dsRNA involved in RNA interference can

> selectively

> silence genes at the read-out or transcription stage

> [6];

> dsRNA species homologous to promoters are involved

> in

> crippling the promoter by methylation (adding methyl

> (-CH3)

> groups) in the region of sequence overlap, so no

> transcription can occur. In other cases, a dsRNA

> resulting

> from a bi-directional transcription of a repeat

> element

> leads to methylation of a nearby histone protein H3

> in

> chromatin, which, too, results in gene silencing.

>

> Transcriptional gene silencing can potentially be

> initiated

> by the dsRNA formed from pairs of transcriptional

> units

> arranged in a tail-to tail orientation (sense

> antisense

> transcription units, SATs). In humans, SATs account

> for most

> overlapping transcriptional units (70%). A recent

> survey

> estimated that there are 1 600 human SATs (or 3 200

> transcription units). When both transcriptional

> units are

> active, formation of dsRNA occurs by default,

> leading to

> modification of the histone protein and gene

> silencing. This

> mechanism is involved in imprinting: the marking of

> genes in

> chromosomes to determine whether they are expressed

> in cell

> clones. _Expression of the gene only occurs when the

> antisense promoter is methylated and inactive.

> Recently, a

> new kind of trans-acting (acting across to different

> parts

> of the genome) RNA was identified in mouse [7]. B2

> RNA

> originates from a short interspersed repetitive

> element

> (SINE) repeated more than 105 copies in the genome

> of

> multicellular plants and animals. They were

> previously

> thought to be molecular parasites with no function.

> However,

> the level of B2 and related RNAs have been found to

> increase

> up to 100-fold in response to environmental stresses

> such as

> heat shock. And B2 RNA is required for the

> concomitant

> inhibition of RNA polymerase II during heat shock,

> by

> interacting directly with the enzyme, preventing it

> from

> working. RNA polymerase II is involved in the

> transcription

> of all protein-coding RNA. So an inhibition of RNA

> polymerase II will decrease the synthesis of many

> proteins.

> A special kind of RNA directed DNA read-out is

> accomplished

> via RNA 'riboswitches' to switch genes off in

> response to

> the concentration of a metabolite in the cell,

> without the

> need for a protein repressor (see Box).

>

>

______

>

> Riboswitch and other RNA regulators

>

> A new molecular switch involves an RNA molecule with

> enzyme

> activity, a ribozyme, which can self-destruct by

> self-

> cleavage [8]. This self-cleavage is accelerated 1

> 000 fold

> in the presence of a small sugar molecule,

> glucosamine-6-

> phosphate, which is generated by the enzyme protein

> encoded

> by a portion of the mRNA downstream from the

> ribozyme

> sequence. So, this simple gene regulatory circuit

> involves

> the mRNA being translated into the enzyme, which

> makes the

> product, glucosamine-6-phospate. As the product

> accumulates,

> it binds to the special catalytic element in the

> mRNA,

> causing it to self-destruct. The region of the mRNA

> that can

> confer this regulatory activity is roughly 75

> nucleotides

> long. When placed upstream of an un-related reporter

> gene,

> it also shuts down its _expression, showing that this

> active

> RNA element is transplantable. A particular group of

>

> ribozymes forms a pocket that binds guanosine

> monophosate,

> one of the four building blocks of RNA. A specific

> region of

> the RNA from the Human Immunodeficiency Virus (HIV)

> binds a

> derivative of the amino acid arginine. Short (<100

> nucleotide) RNA aptamers (DNA or RNA molecules that

> bind

> other molecules) have been identified that

> specifically bind

> everything, from hydrophobic (water-hating) amino

> acids to

> small organic molecules and metal ions. An RNA

> aptamer can

> even distinguish the plant alkaloid theophylline

> from the

> closely related molecule caffeine. Aptamers found

> within

> some natural mRNAs bind small molecules as part of

> their

> gene-regulatory feedback circuits. In the E. coli

> bacterium,

> coenzyme B12 binds directly to, and thereby

> represses

> translation of, the mRNA coding for the protein that

>

> transports its precursor, cobalamin. In Bacillus

> species,

> the synthesis of thiamine and riboflavin involves

> discrete

> genetic units or operons, controlled by direct

> binding of

> thiamine pyrophospate and flavin mononucleotide to

> leader

> sequences of the corresponding mRNAs, resulting in

> the

> premature termination of transcription. Several

> research

> groups had previously engineered artificial

> riboswitches

> that accomplish exactly the same task, that is,

> induce

> ribozyme-mediated cleavage of the RNA on binding

> small

> molecules, before these were discovered in nature.

>

______

>

> RNA splicing

>

> It is estimated that 64% of the genes in the human

> genome is

> interrupted [9]; i.e., the coding regions exist in

> short

> stretches (exons) interrupted by long non-coding

> stretches

> (introns). After the entire sequence is transcribed

> into

> RNA, the non-coding stretches are spliced out,

> leaving the

> coding sequence. However, different exons can be

> spliced

> together, and the borders between the exons and

> introns can

> themselves be shifted. Alternative splicing

> multiplies the

> number of different proteins that can be obtained

> from a

> single gene. This is a case of extensive cutting and

> pasting

> of the genetic text to suit the occasion. The

> fruitfly gene

> Dscam (homologue of the Down syndrome cell adhesion

> molecule) codes for a cell-surface protein essential

> for the

> development of the fruitfly's brain. It has so many

> exons

> that a total of 38 016 possible alternative splice

> forms

> could be generated. Geneticists from the Whitehead

> Institute

> for Biomedical Research, Cambridge, Massachusetts in

> the

> United States analysed the splice forms expressed by

>

> different cell types and by individual cells, and

> found that

> the choice of splice variants is regulated both

> spatially

> and temporally [10].

>

> Different subtypes of photoreceptor cells express

> broad yet

> distinctive spectra of Dscam splice forms.

> Individual

> photoreceptor cells express about 14-50 splice forms

> chosen

> from the spectrum of thousands distinctive of its

> cell type.

> Thus, the repertoire of each cell is different from

> those of

> its neighbours. The complexity does not end there.

> Not only

> are different splice variants obtained from the same

> primary

> transcript, trans-splicing between different primary

>

> transcripts can also take place [11], multiplying

> the

> combinatorial possibilities of proteins available.

>

> There's increasing evidence that genomic variants in

> both

> coding and non-coding sequences in genes can have

> unexpected

> deleterious effects on the splicing of gene

> transcripts

> [12]. Even synonymous base substitutions (those that

> do not

> change the amino acid sequence of the encoded

> protein) and

> sequence changes within the introns can affect

> splicing and

> cause diseases. RNA-directed rewriting of RNA

>

> Some nucleotides are deleted during splicing and

> others

> changed by editing. Around 41 to 60% of mouse

> multi-exon

> genes generate alternatively spliced transcripts,

> the

> frequency of edited transcripts is unknown. These

> processes

> generate new sequences not found in the gene.

> Trypanosomes

> show the importance of RNA rewriting. Their survival

> depends

> on editing defective mitochondrial transcripts using

> trans-

> encoded RNA sequences to guide insertion and

> deletion of

> uridine bases. The rewriting of RNA restores the

> correct

> reading frame, allowing the production of functional

> gene

> products. RNA guides are also used to direct

> rewriting of

> RNA during editing and splicing of pre-mRNA. In some

> cases,

> editing creates splice sites and in others splicing

> prevents

> editing.

>

> Rewriting of RNA is associated with a high turnover

> of

> transcripts. Of all the RNA transcribed in the human

>

> nucleus, only about 5% enters the cytoplasm Quality

> control

> mechanisms dispose of incompletely or improperly

> processes

> messages encoding flawed proteins.

>

> RNA-directed rewriting of DNA

>

> Genomes can be rewritten using reverse transcription

> to

> record elements of successful 'ribotypes'

> (combination of

> RNAs). Around 45% of the human genome is derived

> from

> retrotransposition. RNA-directed rewriting of DNA

> also has

> an essential role in maintaining genome stability.

> Telomerase is a reverse transcriptase that uses an

> RNA guide

> to rewrite the ends of chromosomes (telomeres) and

> prevent

> their loss, which is important for maintaining the

> stability

> of the genome.. Coordination of information

>

> In each ribotype, only specific transcripts are

> produced and

> particular mRNAs translated. These outcomes are

> achieved by

> 'coRNAs' that coordinate the action of highly

> conserved

> pathways. An RNA product from one processing event

> may

> regulate a downstream event, making the second

> outcome

> contingent on the first. For example, a miRNA

> encoded in an

> intron would only be expressed when the host gene is

>

> transcribed. CoRNA may facilitate coordination of

> pathways

> by interacting with sequence motifs shared by a

> number of

> targets. Evolution of rule sets requires creation of

> new

> coRNAs, possibly by duplication and mutation. New

> coRNAS

> would result in assembly of new regulatory complexes

> on

> conserved DNA elements, new patterns of gene

> _expression

> during development.

>

> Replication of ribotypes

>

> Both genetic modification, involving changes in DNA,

> and

> epigenetic modifications, such as DNA methylation

> and

> histone acetylation, can be inherited. For example,

> imprinting is determined by the parent of origin of

> a

> chromosome, which means that at some point maternal

> and

> paternal chromosomes are marked so that they can be

> distinguished during embryonic development.

> Methylation may

> undergo variable erasure during primordial germ cell

>

> development, producing epigenetic mosaic

> individuals. The

> persistence of such epigenetic marks is relevant to

> the

> origin of complex diseases. Here, the susceptibility

> of

> offspring to disease can depend on whether there is

> maternal

> or paternal history of disease as well as ethnicity.

>

> Transmission of ribotypes also occurs more directly.

> The

> embryo receives RNA from the mother that is

> important in

> specifying cells fate. The foetus is also exposed to

> the

> maternal environment, which can influence the foetal

>

> phenotype. For example, pregnant female mice fed a

> diet rich

> in methyl donors have litters with fewer

> yellow-coloured

> agouti Avy offspring, reflecting enhanced silencing

> of the

> retroviral promoter in this allele (see " Diet

> trumping

> genes " , SiS 20

> http://www.i-sis.org.uk/isisnews/sis20.php).

> In other cases, integration of signals received from

>

> maternal hormones may trigger epigenetic

> modifications that

> alter long-term phenotypic development by modulating

> RNA co-

> regulatory networks. Low birth weight, for example,

> has been

> shown to correlate with lifetime risk of

> cardiovascular

> disease and diabetes mellitus. Recently, it has been

>

> demonstrated that the plasma of pregnant women

> contains

> circulating mRNA originating from the foetus [13],

> which is

> rapidly cleared after delivery. This raises the

> question of

> whether coRNAs secreted by various somatic tissues

> are also

> used to transmit information from mother to foetus,

> a

> serious case of the inheritance of acquired

> characteristics

> not coded in the genome.

>

>

>

>

========================================================

>

> This article can be found on the I-SIS website at

> http://www.i-sis.org.uk/RNASTGT.php

>

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