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Effects of retroelements on gene expression Cordaux & Batzer Nat Rev Genet 10:691, 2009 Gogvadze & Buzdin Cell Mol Life Sci 66:3727, 2009 Retrotransposon cis-elements can cause interference with expression of host gene Potential outcomes if L1 located within an intron of gene X? Kines & Belancio Frontiers Biosc 17:1329, 2012 “Intronic retroelements: not just “speed bumps” for RNA polymerase II” ~ 7000 full-length human L1s are “nearly equally distributed between intragenic and intergenic regions” - but “only ~ 100 copies of autonomous & retrotranspositionally competent L1 elements/ genome” ”approximately half of one million Alus are located in introns of genes” Kaer & Speek Mob Gen Elem 23:154, 2012 How to determine if there are bidirectional transcripts & to map them? Primer for cDNA synthesis to test polI type Lunyak et al. Science 317:248, 2007 “Expression of P1-LINE RNA in adult rat tissues” Probe: 32P-labeled 2.8 kb P1-LINE DNA. + lane: 100 pg of unlabelled probe. Panels A & B: RNA from 2 different rats (data for 2 more rats in Suppl Fig). “Some distinct transcripts are marked with arrows”. NTC: no template (1st strand cDNA) control Observations & possible reasons for apparent discrepancies? Singh &. Rath RNA Biol 9:1380, 2012 Retrotransposon-mediated “gene breaking” - presence of L1 retroelement can provide polyadenylation signal & bidirectional promoter leading to fission of pre-exising gene into two short genes TSD: target site duplication (from staggered cut of DNA when TE integrates into new site) Whelan Genome Res 15:1073, 2005 LTR-retroelements can provide new (alternative ) promoters… Human CYP19 (estrogen biosynthesis enzyme) Kamat Trends Endocrin. Metab. 13:122, 2002 Exon 1.1 Northern blots Exon 1.4 “At least 50% of human-specific HERV-K (HML-2) long terminal repeats serve in vivo as active promoters for host nonrepetitive DNA transcription. Buzdin J. Virol 80:10752, 2006 Van de Lagemaat Trends Genet.19:530, 2003 Microsomal glutathione transferase type gene Bioinformatics: - examined 1176 alternatively spliced internal exons & 4151 constitutively spliced ones - 5.2% of alternatively-spliced category had “significant BLAST hit” to Alu (vs. 0% of other class) - most caused frameshift or premature stop codon “Alu elements have the evolutionary potential to enhance the coding capacity and regulatory versatility of the genome without compromising its integrity” Sorek Genome Res 12:1060, 2002 “The birth of new exons” “Alu-ternative splicing” “... a consensus Alu sequence [in antisense orientation] contains 7 potential 5’ splice (donor) sites and 12 potential 3’ (acceptor) splice sites” . .. and “only a few in sense strand” Sorek RNA 13: 1603, 2007 “Alu capture” generates new N-terminal domain Human tumour necrosis factor receptor (TNFR) gene Alu TNFR “From junk to gene...” Singer J. Mol. Biol. 341:883, 2004 Alu indirect repeats in double-stranded RNA structure can undergo A-to-I editing & generate new splice sites Poniscan Curr Opin Dev Genet 20:190, 2010 and Keren Nature Rev Genet 11:345, 2010 “Alu”strious long ncRNAs and their roles in shortening mRNA half-lives SBS: STAU1binding site SMD: STAU1mediated mRNA decay Intramolecular: Staufen1 protein binds specific ds RNA structure & triggers RNA turnover Observation: when STAU1 downregulated, saw bias for upregulation of mRNAs with Alu sequences in 3‘UTR In silico search of expression databases identified lncRNAs (polyadenylated & located in cytoplasm) capable of annealing with these 3‘UTR-located Alu sequences… Led to intermolecular model How could you test this model? Gong & Maquat Cell Cycle 10:12, 2011 LTR-retroelements can contribute to tissue-specific splicing variants Positions of LTR-retroelements present in Wx gene in different maize mutants Wx encodes enzyme for amylose biosynthesis in endosperm & pollen Model consistent with these observations? Marillonnet Plant Cell 9: 967, 1997 Marillonnet Plant Cell 9: 967, 1997 ZmDB Mutant Phenotype Browser * Asterisks denote in-frame stop codons Impact on wx gene product? Northern blot analysis Endosperm RNA Probes: How do you interpret these results? Pollen RNA Marillonnet Plant Cell 9: 967, 1997 RT-PCR analysis RNase protection assay “401 nt and 306 nt protected products can’t be explained by alternative splicing” E = endosperm P = pollen Marillonnet Plant Cell 9: 967, 1997 Retroelements as antisense regulators of host gene transcription Chimpanzee lacks LTR-HERV retroelement in intron of SLC4A8 or IFT172 gene Gogvadze et al. J. Virol. 83:6098, 2009 Gogvadze et al. J. Virol. 83:6098, 2009 Control of heat shock response by SINE RNAs Poniscan Curr Opin Dev Genet 20:190, 2010 There are 2 forms of Alu RNA Full-length & scAlu (short cytoplasmic form, which lacks the right arm RA) How to determine which region(s) of Alu RNA binds RNA pol II (for transcription repression)? Products detected by RT-PCR? - look too discrete for northerns unless “run-off” transcription used (linear template) Mariner Mol Cell 29:499,2008 Impact of retrotransposons on human gene expression Cordaux & Batzer Nat Rev Genet 10:691, 2009