BACE1-AS lncRNA mediating microRNA function

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Faghihi, M A et al ., 2010  reported that BACE1-antisense prevents miRNA-induced repression of BACE1 mRNA by masking the binding site for miR-485-5p. Indeed, miR-485-5p and BACE1-antisense compete for binding within the same region in the open reading frame of the BACE1 mRNA (see the figure below)

Screenshot from 2013-06-20 01:08:56

In order to find confirm if the BACE1-AS competes with th microRNA, the group performed RT-PCR on the RNA samples from different tissues of human and mouse and found that the expression of BACE1, BACE1-AS and mir-485-5p was higher in brain than any other tissue. and since BACE1 gene is know to be involved in Alzheimer’s disease the group examined second set of RNA samples from control subjects and individuals with Alzheimer’s disease.

Initially, they examined the parietal lobe and cerebellum of 5 subjects with Alzheimer’s disease and 5 normal elderly individuals (20 samples total). BACE1-AS, and to a lesser degree BACE1, transcripts were up-regulated in Alzheimer’s disease patients compared to control individuals and miR-485-5p was down-regulated by 30% in parietal lobe and close to 60% in cerebellum of Alzheimer’s disease patients

Screenshot from 2013-06-20 07:44:31

Later they also examined a second set of RNA samples from 35 Alzheimer’s disease and 35 control individuals since samples were not available for all regions, they examined for only few regions and found consistent results as there previous works.

BACE1-AS transcript concentrations were up-regulated in all four regions tested. To a lesser degree, BACE1 transcripts were up-regulated in entorhinal cortex as well as in superior frontal gyrus. On the other hand, miR-485-5p was significantly reduced in entorhinal cortex and hippocampus. However, miR-485-5p was not significantly altered in cerebellum and superior frontal gyrus. The difference between miR-485-5p expressions in cerebellum of the two sets of RNA samples can conceivably be explained by the relatively high variability among human samples. (see the figure below)

Screenshot from 2013-06-20 07:53:32

They conclude demonstrating the potential competition between microRNA and antisence lncRNA for a binding site in the sixth exon of BACE1 gene and hence regulating its level. This study uncovers yet another post-transcriptional mode of gene regulation in which lncRNAs are involved.

Later in 2012 Keniry et al., demonstrated similar post-transcriptional regulation by another lncRNA (h19 lincRNA)  which included miR-675.

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lincRNA-p21

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Yoon J H et al., 2012 uncovered new role of lincRNA as a post-transcriptional inhibitor of translation. They propose that HuR controls translation of a subset of target mRNAs by influencing lincRNA-p21 levels.

  • HuR (human antigen R) or ELAV-like protein 1 is a protein that in humans is encoded by the ELAVL1 gene. The protein encoded by this gene is a member of the ELAVL protein family. This encoded protein contains 3 RNA-binding domains and binds cis-acting AU-rich elements. It stabilizes mRNAs and thereby regulates gene expression

An association between HuR with lincRNA-p21 was detected using the RNP immunoprecipitation (RIP) assay. RIP is an antibody-based technique used to map RNA–protein interactions in vivo by immunoprecipitating the RNA binding protein of interest together with its associated RNA and allows identification of bound transcripts. RIP precipitates a specific RNA binding protein (RBP) and associated RNA (mRNAs, non coding RNAs, viral RNAs) that can be detected by real- time PCR, microarrays or e.g. sequencing. (in this case IP reactions were carried out using HeLa cell lysates and anti-HuR antibody)

The group propose that in the presence of HuR, lincRNA-p21 is unstable through the recruitment of let-7/Ago2. HuR then promotes the translation of targets CTNNB1 and JUNB mRNAs by favoring their association with polysomes and in the absence of HuR, lincRNA-p21 is stable and accumulates, and Rck promotes the association of lincRNA-p21 with CTNNB1 and JUNB mRNAs, repressing their translation through a mechanism that includes reduced polysome sizes and in addition, base-pair interactions of lincRNA-p21 with target mRNAs may result in ribosome ‘drop-off’. In sum, HuR-dependent translation activation requires rapid degradation of lincRNA-p21 in order to prevent the recruitment of translation repressors onto target mRNAs.

Screenshot - 06192013 - 01:08:03 PM

BACE1-AS lncRNA

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Beta-secretase 1 (BACE1) also known as beta-site APP cleaving enzyme 1 (beta-site amyloid precursor protein cleaving enzyme 1), memapsin-2 (membrane-associated aspartic protease 2), and aspartyl protease 2 (ASP2) is an enzyme that in humans is encoded by the BACE1 gene.  β-Secretase is an aspartic-acid protease important in the formation of myelin sheaths in peripheral nerve cells.

Faghihi, M. A et al., 2008 identified a conserved noncoding antisense transcript for β-secretase-1 (BACE1), a crucial enzyme in Alzheimer’s disease pathophysiology. The BACE1-antisense transcript (BACE1-AS) regulates BACE1 mRNA and subsequently BACE1 protein expression in vitro and in vivo. In order to find the regulatory effects of BACE1-AS the group performed the experiments on SH-SY5Y cell lines and transacted the cell lines by small interfering RNAs (siRNAs) targeting on the non-overlapping regions of BACE1-AS transcript and the observed surprising results. Along with the knockdown of the BACE1-AS, BACE1 mRNA were also knocked down confirming the regulatory effects on the BACE1-AS on BACE1 gene. (see figure)

Screenshot - 06172013 - 07:11:44 PM

They repeated the similar experiments on two other cell lines and each time with three different siRNAs, all of them gave the similar outcome proving the concordant regulation of BACE1-AS on BACE1 gene.

LncRNA MALTAT1 Regulates alternative splicing

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MALTAT1 is the lncRNA which was found to regulate the alternative splicing of pre-mRNAs (Prasanth KV et al., 2010). This lncRNA interacts with the SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the alternative splicing of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins.

The group demonstrated that MALAT1 localizes to nuclear speckles and interacts with several pre-mRNA splicing factors also the depletion of the same results in an increase in the dephosphorylated pool of SR proteins that display a more homogeneous nuclear distribution. Since the SRSF2 mAb primarily recognizes phosphorylated SR proteins, it likely detects a steady-state pool of residual phosphorylated SR proteins present in MALAT1- depleted speckles (can be seen in figure below). Hyper-Phosphorelated SR domains influence the binding of SR proteins to pre-mRNA and regulate splice site selection by dictating protein-protein and protein-RNA interactions within the spliceosome.

Screenshot - 06172013 - 02:31:46 PM

First-Ever report on lncRNA in 1990 (mouse H19)

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The mouse gene H19 is the first reported as Long non-coding RNA by (Brannan C.I et al., 1990), The group showed despite the fact that this gene is transcribed by RNA polymerase-II and is spliced and polyadenylated like any other mRNA, H19 RNA is “not a classical mRNA”. Instead, the product of this unusual gene may be an RNA molecule.

H19 gene was first identified in a screen of fetal liver cDNA library to find the genes that were coordinately regulated with the AFP (Alpha Feto Protein) gene. The AFP gene is located on the q arm of chromosome 4 (4q25). AFP is the most abundant plasma protein found in the human fetus. Plasma levels decrease rapidly after birth but begin decreasing prenatally starting at the end of the first trimester. Normal adult levels are usually achieved by the age of 8 to 12 months.

Pachnis V et al. 1984, found that the levels of alpha fetoprotein in mice are determined by at least  two trans-acting, unlinked genes, raf and rif. raf determines the basal levels of a-fetoprotein mRNA in adult mice, while Rif determines its degree of inducibility during liver regeneration. To determine whether these regulatory loci affect other structural genes, the screened a murine fetal liver cDNA library for clones containing mRNA sequences that decrease after birth. One such clone, termed pHl9, was identified, and its mRNA was shown to be under the control of both raf and Rif. The single-copy gene for H19 “mRNA” was localized to chromosome 7.

In 1990 Brannan C.I et al., found H19 gene was expressed at high levels in fetal and neonatal liver, and its 600-fold repression in the adult was under the control of raf. Sequence analysis of the single-copy H19 gene revealed the presence of multiple small open reading frames (ORFs), none of which spanned more than two of its five exons. The largest of these, called ORF5 was located entirely within the first exon and could potentially encode a 132-amino-acid protein. This ORF begins 680 bases downstream of the cap site and is preceded by four small ORFs, the largest of which could encode a protein of 27 amino acids. As there was no precedent for such a gene organization and hence the group decided to investigate.

And found H19 It shares features common to other mRNAs: it is transcribed by RNA polymerase II, processed by RNA splicing, and polyadenylated. Unlike mRNAs, it is not associated with ribosomes in any tissue or at any stage in development that was examined. They concluded from the findings that the RNA is never translated and even if H19 RNA functions as a mRNA, then it must be under stringent translational control. However they considered H19 gene functions as an RNA molecules, hence it was the first large non-coding RNA.

Shortly afterwords another non-coding (second) was found to be expressed exclusively from the inactive X chromosome and later demonstrated to be required for X inactivation in mammals and termed as XIST lncRNA.

COOLAIR lncRNA

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COOLAIR: Cold Induced Long Antisense Intragenic RNA (Swiezewski et al., 2009) is a transcript antisence to FLC gene which is responsible in regulating the flowering time in Arabidopsis plant.

Plants like Arabidopsis when is in a prolonged cold condition shows the epigenetical silencing of FLC (Flowering Locus C) in a Polycomb mediated process called vernalization (De Lucia, F. et al. 2008). Swiezewski and group could show that upregulation of long non-coding antisense transcripts covers the entire FLC locus which may be part of the cold-sensing mechanism. Induction of these antisense transcripts occurs earlier than, and is independent of, other vernalization markers and coincides with a reduction in sense transcription. This antisense mediated gene silencing involves the Polycomb protein machinery.

other useful links:

  1. Robert letswaart 2012 http://www.sciencedirect.com/science/article/pii/S0168952512000911

HOXAIR lincRNA

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HOXAIR: HOX Antisense Intergenic RNA (John L. Rinn 2007) is transcribed in antisense manner with respect to the canonical HOXC genes. Molecular cloning and Northern blot analysis confirmed that HOTAIR is a 2158 nucleotide, spliced, and polyadenylated transcript.

HOXAIR was found to suppress the HOXD complex genes (in trans). This trans activity of HOXAIR was confirmed by the study conducted by Rinn et al., in which the depletion of HOTAIR by RNA interference (siRNA mediated) lead to dramatic transcriptional activation of the HOXD locus on chromosome 2 spanning over 40 Kb, including HOXD8, HOXD9, HOXD10, HOXD11, and multiple ncRNAs. To ensure that this was not an off-target effect of RNA interference, four independent siRNA sequences targeting HOTAIR. Each siRNA depleted HOTAIR ncRNA and led to concomitant HOXD10 activation as determined by quantitative RT-PCR. These observations indicate that HOTAIR ncRNA is required to maintain a transcriptionally silent chromosomal domain in trans on the HOXD locus.

Molecular Mechanism

It was found that HOTAIR enhances the PRC2 (Poly-comb protein complex 2) activity at HOXD locus. To investigate the molecular mechanisms involved in the HOTAIR dependant silencing of the HOXD locus, the group performed chromatin immunoprecipitation to interrogate changes to the HOXD chromatin structure upon depletion of HOTAIR.

  • Polycomb Repressive Complex 2 (PRC2), comprised of H3K27 histone methyl transferase (HMTase) EZH2 and core components Suz12 and EED, initiates this histone modification and subsequently Polycomb Repressive Complex 1 (PRC1) maintains this modification and promotes chromatin compaction (reviewed by (Sparmann and van Lohuizen, 2006)
  • Chromatin Immunoprecipitation (ChIP) is a type of immunoprecipitation experimental technique used to investigate the interaction between proteins and DNA in the cell

Previous ChIP-chip experiments indicated that in primary foreskin fibroblasts, the entire HOXD locus was occupied by both Suz12 and H3K27me3. Depletion of HOTAIR followed by ChIP-chip revealed substantial and global loss of H3K27Me3 occupancy over the HOXD locus, with the greatest loss residing in the intergenic region between HOXD4 and HOXD8. HOTAIR depletion also led to a modest but consistent loss of Suz12 occupancy of the HOXD locus. Importantly, occupancy of H3K27me3 and Suz12 across the silent HOXB locus was not affected by HOTAIR depletion in these cells. These results suggest that HOTAIR is selectively required to target PRC2 occupancy and activity to silence transcription of the HOXD locus.

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