Saturday, 22 December 2012

RNA interference

Long arm of long noncoding RNAs: roles as sensors regulating gene transcriptional programs by Wang et al
Subcellular localisation
Most mRNAs localise to cytoplasm after processing. Mostl ong ncRNAs are permanently localised in nucleus.

Most are transcribed by RNAP II.  Transcription is regulated with basic rules. Histone H3K4me3 methylation mark at promoters and H3K36me3 marks in transcription body.

Long ncRNAs are often spliced.

Mechanisms mediating transcriptional regulation of long ncRNAs
Epigenetic regulation eg histone nad DNA methylation and post-translational modifications and remodelling complexes. Long ncRNas can affect loading of GTFs and polymerase and modulate activities of spec TFs.

Long noncoding RNAs: functional surprises from RNA world by Wilusz

When act of ncRNA transcription alone may be enough
ACt of transcribing ncRNAs can affect ability of nearby genes to be expressed. Transcription of ncRN A across promoter of a downstream gene can interfere with TF binding and prevent expression. Transcriptional interference regulates developmental decisions eg where Hox genes are expressed.

Transcription of ncRNAs can induce histone mofifications. These repres transcription inititation of overlapping genes.

Noncoding transcription can induce formation of heterochromatin at p15 tumour suppressor gene locus. It persisted after noncoding transcription was turned off.

Long ncRNAs target proteins to spec genomic loci to affect transcription patterns
PcG proteins bind and silence genes. ncRNAs may target PcG proteins to spec genomic locations.

Long ncRNAs may assure that only one of 2 parental alleles are expressed. In mouse placenta, Airn ncRNA is required for paternal silencing in cis. Appears to coat imprinted locus on paternal Nchromosome. It accumulates at Slc22a3 promoter. Air interacts with a methyltransferase, methylating and silencing paternal Slc22a3 promoter.

Long ncRNA modulate activity of protein-binding partners
RNAs influence activity and localisation of proteins they bind. Eg coactivators of proteins in transcriptional regulation. Evf2 ncRNA forms a complex with Dlx2 protein. Dlx2 acts as an enhnacer only when Evf2 ncRNA is present.

ncRNA HSR1 enables HSF1 to induce expression of heat shock proteins.

Long ncRNAs as precursors for small RNAs
Long ncRNAs may be posttranscriptionally processed to yield small RNas wit a 5' cap structure.

No-nonsense functions for long noncoding RNAs by Nagano
Scaffolds for histone modifiers
lcRNA can bind chromatin modification complexes. RNAs may recruit PRC2 to sites of synthesis. HOTAIR lncRNA is necessary for PRC2 occupancy, H3K27me3 and silencing of HoxD locus.

lncNAs as nucleators of nuclear structures
Paraspeckles are discrete ribonucleoprotein bodies in mammalian cell nuclei  implicated in nuclear retention of hyperedited mRNAs. Mao expressed fluorescently tagged paraspeckle-associated fusion proteins in cells with an inducible Men lncRNA, the RNA component of paraspeckles. Paraspeckle-associated proteins were recruited and assembled on Men lncRNAs as they were transcribed. These assembled structures persisted near the nuclear site of transcription.

The X as model for RNA's niche in epigenomic regulation by Lee
During X chomosome inactivation (XCI), Xi is distringuished from Xa by enrichment in Polycomb proteins, HP1 and chromatin modifications.

In 5' end of Xist locus is a motif Repeat Am which encodes a transcription unit called RepA. RepA RNA binds polycomb proteins. It recruits them to Xic. This activates Xist expression and initiation of XCI.

Actions of RepA and Xist are controlled by Tsix, a ncRNA antisense to both RNAs.

In undifferentiated cells (pre-XCI) Tsix is expressed on both Xs. Xist RNA is expressed at low levels. At cell differentiation, Tsix persists only on future Xa. Tsix RNA prevents upregulation of Xist on Xa.

Downregulation of Tsix on Xi allows Xist transcativation.

Whether Tsix RNA persists during cell differentiation is controlled by upstream lcus Xite. Xite has a developmentally regulated enhancer. Enhancer maintains expression of Tsix on Xa and prevents inactivation of Xa.

Polycomb proteins targeted and regulated by RNA
During silencing step, recuitmemnt of PCR2. PCR2 is recruited to X almost immediately after Xist RNA accumulation. PCR2 is brough by RepA RNA. This causes heterochromatic state.

Tsix can also bind PRC2. Tsix may be competitive inhibitor for PRC2.

In undifferentiated ES cells and future Xa, high levels of Tsix RNA prevent XCI by interefering with RepA-PRC2 complex.

When Tsix is downregulated on future Xi, RepA RNA productively engaged PRC2, target polycomb actvivity to Xist promoter and triggers uprgulation of Xist RNA by trimethylating H3K27. This creates heterochromatin at 5' end of Xist.

Xist binds PRC2. This spreads PRC2 and its H3K27trimethylase activity along X to initiate chromosome wide silencing.

Gene silencing in X chromosome inactivation: advances in understanding facultative heterochromatin formation by Wutz
Nuclear scaffold protein SAF-A is enriched on Xi in female somatic cells. Its localisation to Xi requires SAF-A/RNA binding domain and Xist expression.  Binding of Xist RNA to SAF-A has been obsered.  This indicates that Xist interacts with nuclear scaffold to localise in cis over X chromosome.

Xist binding overlaps with sequences enriched for genomic repeats. They are distinct from gene- containing regions of X chromosome which are at periphery of X chromoosme and do not overlap with most of Xist binding sites.

Formation of a repressionve compartment
After Xist localisation over Xi, TF initiation factors, RNAP IIand splicing factors are depletd from Xist-cvered chromatin domain. Transcripion machinery may be exluded from Xist domain. Causing transcriptionally inactive and repressive compartment   Nascent RNA is depleted. Absence of histone modifications associated with active genes eg histone acetylation or methylation oh H3 at Lysine 3 (H3K4me).

Chromatin in Xist domain is modified by activity of PcG complexes. 2 catalytically active PcG complexes are enriched at Xi: Polycomb repressive complex 1 (PRC1) which catalyses ubiquitylation of histone 2A at lysine 119 and PRC2 which catalyses trimethylation of H3K27.

To initiate gene silencing, Xist repeat A is required. It is a conserved RNA sequence motif at 5'end of Xist.

Most chromosomal DNA is composed of genomic repeats and noncoding sequences. Genes are at periphery of chromosome territory. Active genes protrude into nuclear space on chromatin loops. Xist localises to centre of CT and triggers formation of a repressive compartment. This excludes transcription machinery. PcG modified chromatin.  Silencing factors eg ATB1 (AT-rich sequence binding protein 1) are present. SATB1 binding to chromatin loops may make gene susecptible to repression by Xist. This involves repeat A region of Xist RNA. Silent genes associate with repressive compartment.

Stability and maintenance
During development  Initially silenced Xi changes until a stably inactivated Xi in female somatic cells which do not require Xist to maintain inactivation.  In mouse ESCs, switch from Xist dependent, reversible inactivation to Xist-independent silencing.

Stabilisation in embryonic lineages
DNA methylation of X-linked gene promoters is associated with Xi repression. In somatic cells, Xi is hypomethylated in gene poor regiomns. Hypermethylated in gene rich regions relative to active X chromosome.

Dnmt1 is required for stable maintenance of XCI in embryonic development. In mice Dnmt1 mutation causes failure in maintenace of gene silencing on Xi. DNa methylation is important to stabilise XCI. DNA methylation on Xi is preferentially enriched on gene promoters. OVerall sequences are less methylation on Xi compared to active X

Stabilisation in extra-embryonic lineages
Mutations in Dnmt1 and Smchd1 are dispensable for imprinted XCI in extra-embryonic tissues. Other pathwats apart from DNA methlation lock in XCI in lineage specific manner. Mutation of PcG group gene extra-embryonic development (Eed) in mice causes female-spec phenotype in trophoblast develpoment. XCI may fail to be imprinted in extra-embryonic tissues.  Reporter gene on paternally inherited Xi is reactivated.

Xi facultative heterochromatin in somatic cells
GEne silencing on Xi in somatic cells is stable.  Xi in female somaitc ells has spec chromatin composition that distringuishes it from other types of heterochromatin eg pericentric heterochromatin or nucloelar heterochromatin.  Spec proteins are enriched on Xi. Polycomb proteins recruited to Xi catalse H3K27me/  USe ChIP or ChIP-seq.

Reactivation of inactive X chromosome
In female somatic cells, genes on Xi are stably repressed, unlike early embryo. Reactivation of Xi accompanies development of germ line. Loss of Xist expression during primordial germ cell formation. Xi-linked genes are reactivated gradually during germ cell migration. Female germ cells have 2 active X chromosomes during oogenesis.

This is maintained in mouse ESCs. Fusion of female somatic cells with mouse ESCs tgriggers reactivation of Xi from somatic fusion partner. ESCs cam reverse epigeenetic modifications that stabilise Xi in somatic cells.

reactopm pf Xi with reprogramming of female mouse somatic ells to pluripotent state.

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