ANTISENSE TRANSCRIPT & HUMAN DISEASE

1. ANTISENSE TRANSCRIPT & HUMAN DISEASE Huong Le Dept of Molecular & Clinical Genetics, RPA Hospital

2. Natural antisense transcriptsNATs • To understand biological process, it is essential to understand the regulatory network of genes • Regulation of gene expression is well known to be conducted by transcription factors however the non coding RNAs include natural antisense transcripts (NATs) also have a similar role in this function • The antisense mRNA regulates the expression level of the sense mRNA in a pair

3. NAT types • cis-NATs • Composed of a pair of overlapping transcripts that are transcribed from the same genomic locus of the sense strand but in the opposite direction • trans-NATs • A pair of overlapping transcripts that originated from the different genomic locus of the sense strand but in the opposite direction Small trans-NAT can be • microRNA • siRNA S AS

4. Classification of cis-NAT • Based on their positions in the genome Genetics 2007;176: 1299–1306

5. Functional antisense transcripts • Because mRNA is a ssRNA and therefore binding of antisense strand complementary may alter gene expression at many levels including • Transcription exclusion • Genomic imprinting • DNA methylation • X- inactivation • Alternative splicing • RNA interference (RNAi)

6. Regulation of gene expression by cis-NAT • 3 models proposed for regulation of gene expression by cis-NAT • Cis-NAT form a duplex complementary with the sense transcript lead to inhibition functional of mRNA and protein synthesis • Epigenetic regulation (methylation of promoters of sense transcripts and inhibit the transcription of sense strands • Transcriptional collision (RNA polymerase bind to promoters of genes encoding sense and cis-NAT transcripts. The RNA polymerase was clash in the the overlapping region and inhibit their transcription.

7. www.drugdiscoverytoday.com

8. Imprinting gene • Two parental genes or alleles are expressed differently • In most case one allele is silent throughout the development. This allele remains in the memory of parental origin “imprinted gene” • Part of imprinted marks is DNA methylation (an addition of methyl group covalently bind to cytosine residue in the CpG islands (IC regions)

9. Imprinting defects in 15q11-q13 • This chromosomal region is affected in the Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) • PWS cause by the lost function of paternal expressed genes • AS cause by the lost function of maternal expressed gene (UBE3A)

10. Clinical Chemistry. 2006;52:1276-1283 • Prader–Willi syndrome (PWS) and Angelman syndrome (AS) are the most common genetic disorders involving non-Mendelian inheritance in the form of genomic imprinting • PWS occurs in 1 of every 15 000 live births caused by the loss of expression of paternal expressed genes in the region of 15q11.2-q13 • severe hypotonia and feeding difficulties • excessive eating and gradual development of morbid obesity • 70% have a 15q11.2-q13 deletion on the paternally inherited chromosome 15 • 25% have maternal uniparental disomy (UPD • <5% have an imprinting center sequence variant,

11. Clinical Chemistry. 2006;52:1276-1283 • AS is a neurogenic disorder caused by the loss of function of the imprinted ubiquitin protein ligase E3A (UBE3A) gene in 15q11.2-13 • severe mental retardation with absence of speech, microcephaly • inappropriate laughter, seizures, and a stiff gait. • 70% of individuals with AS have a 15q11.2-q13 deletion of the maternal-origin chromosome • 11% have a sequence variant in UBE3A, • 7% of AS patients have paternal UPD, • 3% have an imprinting center sequence variant

12. Classifications of PWS / AS Molecular Human Reproduction 1997;3:321-332

13. UBE3A and UBE3A antisense transcripts • UBE3A gene (ubiquitin protein ligase E3A ) • Predominantly express from maternal chromosome 15 in brain but shows bi-allelic expression in fibroblast and lymphoblast • Difference for most paternal expressed genes and UBE3A due to region of differential methylation exists between maternal and paternal alleles

14. Rougeulle et al. 1998 reported the existence of an antisense transcript to the human UBE3A gene and suggested that maternal only expression of UBE3A may results from tissue specific expression Chamberlain and Brannan (2001) found that paternal transmission of the PWS-IC deletion results in this lack UBE3A antisense transcript And therefore this antisense transcript belong to the class of imprinted genes and have shown to be expressed exclusively from paternal allele and to be dependent on the PWS-IC for expression The repress expression of paternal allele of the UBE3A gene occur as a indirect result of expression of the UBE3A antisense transcript

15. PWS / AS genes

16. SnoRNAs • Small nucleolar RNAs (snoRNAs) • Class of small RNA guide the chemical modifications (Methylation and Pseudouridylation) of rRNAs and other RNA genes • Evidence shown that the snoRNA HBII-52 regulates alternative splicing of the serotonin receptor 2C (Kishore S et al. 2006) • SnoRNAs guide families include • C/D box snoRNAs guide methylation • H/ACA box snoRNAs guide pseudouridylation

17. SnoRNAs Nature Genetics40, 688 - 689 (2008)

18. LUC7L antisense and expression of HBA2 Tufarelli (Nature Genetics 2003;34: 157-165)

19. LUC7L antisense and expression of HBA2 Nature Genetics 2003;34: 125-126

20. Silencing by antisense RNA • How does the antisense RNA lead to methylation and promoter silencing / imprinting? • Possible mechanisms • Overlapping the promoter of imprinted gene may interfere with transcription of the gene by direct competition / methylation of the CpG island in the promoter region • RNA interference related mechanism lead to block the translation or lead to transcript degradation • Trigger silencing • promoter methylation by double stranded RNAs • formation of heterochromatin