Ube3a-ATS

''UBE3A-ATS/Ube3a-ATS'' (human/mouse), otherwise known as ubiquitin ligase ''E3A-ATS'', is the name for the antisense DNA strand that is transcribed as part of a larger transcript called ''LNCAT'' (large non-coding antisense transcript) at the ''Ube3a'' locus. The ''Ube3a'' locus is imprinted and in the central nervous system expressed only from the maternal allele. Silencing of ''Ube3a'' on the paternal allele is thought to occur through the ''Ube3a-ATS'' part of ''LNCAT'', since non-coding antisense transcripts are often found at imprinted loci. The deletion and/or mutation of ''Ube3a'' on the maternal chromosome causes Angelman syndrome (AS) and ''Ube3a-ATS'' may prove to be an important aspect in finding a therapy for this disease. While in patients with AS the maternal ''Ube3a'' allele is inactive, the paternal allele is intact but epigenetically silenced. If unsilenced, the paternal allele could be a source of active Ube3a protein in AS patients. Therefore, understanding the mechanisms of how ''Ube3a-ATS'' might be involved in silencing the paternal ''Ube3a'' may lead to new therapies for AS. This possibility has been demonstrated by a recent study where the drug topotecan, administered to mice suffering from AS, activated expression of the paternal ''Ube3a'' gene by lowering the transcription of ''Ube3a-ATS''.

''LNCAT'' organization

The human ''UBE3A-ATS'' is expressed as a part of ''LNCAT'' mainly from the paternal allele in the central nervous system (CNS). The transcript is about 450 kbs long, starts at the U-exons and extends as far as ''UBE3A'' on the opposite strand, possibly beyond. The promoter for '' Snurf/ Snrpn'' and the imprinting center are found in the U-exon region. The promoter region is imperative, as deletion of this area abolishes ''Ube3a-ATS'' transcription. Near the promoter is the PWS-IC and about 35 kbs upstream of the PWS-IC is the AS-IC. These two regions are thought to control the expression of the entire ''LNCAT'' strand. Starting at the promoter, the entire transcript can be transcribed and after transcription is further processed and spliced. Reviewed in ''Trends in Neurosci''.

Located next to the U-exon promoter region is ''Snrpn/Snurf'' which can be alternatively spliced into either ''Snrpn'' or ''Snurf'' in humans (in mice this remains as one bicistronic transcript). ''Snrpn'' codes for a protein of unknown function which localizes to the cell nucleus. ''Snurf'' codes for a small nuclear ribonucleoprotein. While most of these proteins are involved in splicing, the role of this particular protein is not yet known. Downstream from ''Snrpn/Snurf'' and within its introns are sequences for several C/D box snoRNAs. Most C/D box snoRNAs function in non-mRNA methylation. However, recently, one snoRNA on ''Ube3a-ATS'', SNORD 115, has been found to change the alternative splicing of the serotonin receptor 2C pre-mRNA. In addition, this snoRNA has the ability to change the splicing of five different mRNAs. Among the sequences for the snoRNAs is nested IPW (imprinted Prader-Willi), a non-coding region whose deletion is thought to cause Prader-Willi syndrome.

Model systems

The mouse and human ''Ube3a-ATS/Ube3a'' are orthologous and the general organizations of the regions are similar. For example, the mouse locus also contains ''Snurf/Snrpn'', snoRNAs and IPW. The main differences are the locations and the lengths of the ''Ube3a-ATS'' transcripts. The human ''Ube3a/Ube3a-ATS'' is located on chromosome 15, while the mouse ''Ube3a'' is located on chromosome 7. The mouse ''LNCAT'', including ''Ube3a-ATS'', is about 1000 kb long, much longer than the human 450 kb ''LNCAT''. Due to the similar organization of the mouse and human ''LNCAT/Ube3a-ATS'' and the fact that the mouse ''Ube3a'' locus is also imprinted, the mouse is an excellent model system to study imprinting and the interactions between ''Ube3a/Ube3a-ATs''. In addition, mouse neurons continue to retain their imprinting pattern in culture.

Splice variants and locations

While the entire ''LNCAT'' transcript, including the ''Ube3a-ATS'' transcript may be transcribed, it is often spliced to include and exclude a variety of exons. Different splice variants are expressed in different tissue types and situations. For the most part, it is thought that at least some type of ''Ube3a-ATS'' is expressed in CNS cells that are imprinted, such as Purkinje cells and hippocampal neurons. However, there is spatiotemporal regulation of both the downstream and the upstream part of this transcript.Reviewed in ''Dev Bio'' and ''Journal of Neuroscience''.

In mouse embryos, ''Snurf/Snrpn'' exons were detected in blastocysts about 7 days post coitem and continued to be expressed throughout development. The ''Snurf/Snrpn'' exons are restricted to CNS tissue during development, and only later during adulthood are expressed in other tissue. ''Ube3a-ATS'' exons were not detected until 10 days post coitem and their expression was also limited to the CNS during development. In general, ''Ube3a-ATS'' is detected during the initial stages of neurogenesis while ''Snurf/Snrpn'' is expressed in undifferentiated precursor cells and throughout the course of differentiation. There are at least 10 different splice isoforms according to the UCSC genome browser.

According to one study, the splice variant that directly overlaps the ''Ube3a'' is found in the cytoplasm.

Preventing expression on both alleles

A specific imprinting center cluster was thought to control the differential expression of ''Ube3a-ATS'' on the maternal and paternal alleles. There are two regions in the imprinting centers (ICs) that exist associated with AS and PWS- the AS-IC and the PWS-IC. These imprinting centers are control regions that dictate whether surrounding genes and regions can be expressed and are found on both the maternal and paternal alleles. While differential methylation patterns on the maternal and paternal genes are often associated with imprinting, the AS-IC remains unmethylated at both alleles. However, the neighboring PWS-IC is methylated on the maternal allele, but remains unmethylated on the paternal allele.

The PWS-IC is suspected of controlling the expression of ''LNCAT'' and ''Ube3a-ATS''. In mice where the PWS-IC has been deleted, expression of the ''Ube3a-ATS'' is decreased. In the central neural system, ''Ube3a-ATS'' is preferentially expressed from the paternal allele where the PWS-IC is not methylated. On the other hand, on the maternal allele, where the PWS-IC is methylated, ''Ube3a-ATS'' is not expressed, suggesting that the methylation of the PWS-IC somehow prevents ''Ube3a-ATS'' expression. This is supported by several studies where preventing methylation of the PWS-IC by knocking out methyl transferases in embryonic stem cells results in biallelic expression of ''Ube3a-ATS'' and silencing of ''Ube3a'' on the maternal allele.

However, methylation is not the only process involved in preventing the expression of the maternal ''Ube3a-ATS''. It is expected that the imprinting domains interact with other proteins, which contribute to the silencing of ''LNCAT'' and ''Ube3a-ATS'' on the maternal allele. For example, when ''MECP2'' is knocked out, such as in Rett syndrome patients, ''Ube3a-ATS'' is biallelically expressed, decreasing expression of ''Ube3a'' from the maternal allele.

Collision model

Only one RNA polymerase (RNAP) can transcribe along a part of the template at a time. When two RNAPs are transcribing in head on directions, a collision can occur. This can result in stalling of both of the RNAPs, backtracking of one RNAP, or falling off of the template. In this case, the RNAP transcribing the paternal UBE3A-ATS competes with the RNAP transcribing UBE3A and pushes it off of the template, preventing UBE3A transcription and allowing UBE3A-ATS transcription. Arrows show the direction of transcription. Reviewed in ''Trends in Neurosci''.

There are currently three models that explain how the ''Ube3a-ATS'' of ''LNCAT'' silences the paternal ''Ube3a''- the collision model, the RNA-DNA interaction model, and the Double-stranded RNA, double stranded RNA interference model. While these models have not been demonstrated directly for ''Ube3a/Ube3a-ATS'', they are considered plausible based on evidence for the silencing of other natural antisense transcripts by these methods. However, the collision model, due to most recent supporting studies, appears most likely.

The collision model can be thought of as a road wide enough for only one car. A smart car is traveling from one direction, and a plough from the other direction, eventually colliding. After the collision, the plough pushes the smart car backwards, as it continues to travel forward. In the collision model for ''Ube3a/Ube3a-ATS'', RNA polymerases (RNAPs) travel towards each other along the sense and antisense templates during transcription. The sense and antisense templates overlap for ''Ube3a'' and ''Ube3a-ATS''. The two transcription bubbles will collide head-on, and the RNAP transcribing the ''Ube3a-ATS'', being the plough, will push the RNAP transcribing the ''Ube3a'' (smart car), backwards and eventually off of the template. This prevents complete transcription of Ube3a.

The support for this model comes from two recent studies. The first study looked at transcription of genes on sense strands that were overlapped by genes being expressed on the antisense strand. The longer the region of overlap, the less efficient the transcription of the sense strand was, indicating that transcription on one strand interferes with the transcription on the other strand. Another study directly monitored collisions between RNAPs transcribing a template using atomic force microscopy. RNAPs were stalled on DNA fragments and collided with other elongating RNAPs. The images showed stalling of the two RNAPs immediately after the collision, in addition to backtracking of one of the RNAPs.

While these studies have not been performed for ''Ube3a/Ube3a-ATS'', the use of atomic force microscopy to monitor transcription at this locus might provide insight as to how ''Ube3a'' is actually silenced via ''Ube3a-ATS''. Further studies are still very much necessary to confirm these models for ''Ube3a''.

Contradictory studies

While several studies support the idea that ''Ube3a-ATS'' might be involved in paternal ''Ube3a'' silencing, other studies contradict this. One study in particular argues against the in cis silencing of ''Ube3''a by ''Ube3a-ATS''. In this study, when the maternal ''Ube3a'' allele was deleted, an increase in paternal ''Ube3a-ATS'' expression was seen. This suggests that rather than the paternal ''Ube3a-ATS'' controlling paternal ''Ube3a'', the maternal ''Ube3a'' somehow suppresses expression of the paternal ''Ube3a-ATs'', possibly ''in trans'' rather than '' in cis''. An interaction between the maternal and paternal homologous regions of these genes was in fact observed in human and mouse cells during interphase.

One mechanism to explain ''in trans'' silencing includes an interaction between the paternal ''Ube3a-ATS'' RNA and the maternal ''Ube3a'' mRNA. It is possible that the maternal ''Ube3a'' mRNA interacts with the paternal ''Ube3a-ATS'' RNA and decreases the stability of both of these transcripts. When only ''Ube3a-ATS'' is made without ''Ube3a'', the ''Ube3a-ATS'' becomes more stable.

Another study has suggested that ''Ube3a-ATS'' expression does not occur in imprinted regions. ''In situ'' hybridizations did not reveal ''Ube3a-ATS'' in Purkinje cells or hippocampal neurons. However, other upstream exons that correspond to '' Snurf/ Snrpn'' were expressed, indicating that the collision model could still be occurring. Thus further research is still required.

The future

Several studies have attempted to utilize the possibility of controlling ''Ube3a'' expression through ''Ube3a-ATS''. In AS, the paternal PWS-IC is not methylated, supposedly allowing ''Ube3a-ATS'' expression. Therefore, if methylation of the PWS-IC were possible, ''Ube3a-ATS'' transcription could be prohibited, allowing ''Ube3a'' expression from the paternal allele to make up for the lack of expression from the maternal allele. A one year study was performed with several AS patients. These patients were put on methylation promoting diets that consisted of betaine, metafolin, creatine, and vitamin B12 supplements. However, after one year, methylation patterns in these patients did not change.

Another study tested a large library of different drugs, and identified several topoisomerase I and II inhibitors which increased expression of paternal ''Ube3a'' in mouse neurons and mice. Topoisomerase inhibitors are widely used as chemotherapeutics and cause replicating cells to undergo apoptosis by inducing double strand breaks that stall the replication fork. However, their mechanism of action in activating the paternal ''Ube3a'' is not yet known, but may involve transcriptional interference with ''Ube3a-ATS'', as ''Ube3a-ATS'' transcripts decreased after drug treatment. The group specifically chose to study topotecan, which was the most effective at a low nanomolar range and is already Food and Drug Administration approved for treating several types of cancers.

References

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Genes on human chromosome 15
Antisense RNA