LINEs and SINEs ….& towards cancer! Presenter: Manindra Singh

1. LINEs and SINEs ….& towards cancer! Presenter: Manindra Singh Course: MCB 720 (Winter Qt.)

2. Overview LINEs ( Long Interspersed Elements) and SINEs (Short Interspersed Elements) are 2 classes of Non LTR Transposons, also called as nonviral retrotransposons Non-LTRRetrotransposons transpose through an RNA intermediate utilizing a reverse transcriptase and lack flanking terminal repeat sequence (LTRs). LINEs are ~ 6 Kb long, Observed in protozoans, insects, and plants. Abundant in mammalian genomes SINEs are ~ 300 bp long and are found primarily in mammalian DNA

3. LINEs in Human Genome LINE sequences are present at ≈900,000 sites in the human genome. Account for 21 % of total human DNA! Human DNA contains three major families of LINE sequences: L1, L2, and L3 L1, L2 and L3 LINE elements differ in their sequences, but mechanism of transposition is similar, involving an RNA intermediate Only members of the L1 family transpose in the contemporary human genome

4. SINEs in Human Genome Second most abundant class of mobile elements in the human genome SINEs occur at about 1.6 million sites in the human genome, and constitute ≈13 percent of total human DNA Most of the SINE sequences contain a single recognition site for the restriction enzyme AluI(AGCT)and are called Alu elements - present in ~ 1.1 million sites Aluelements are scattered throughout the human genome: between genes, within introns, and in the 3’UTRs of some mRNAs Alu elements exhibit considerable sequence homology with 7SL RNA, a component of the signal-recognition particle in the ER membrane

5. General Structure of a LINE • LINEs are flanked by short direct repeats of variable length and contain two long open reading frames (ORFs) • ORF1, ≈1 kb in length, encodes an RNA-binding protein • ORF2, ≈4 kb in length, encodes a bifunctional protein with reverse transcriptase and DNA endonuclease activity from Lodishet al., Molecular Cell Biology, 5thed. Fig 10-15

6. Proposed Mechanism of LINE Transposition Move by ‘copy & paste’ mechanism LINE mRNA is transcribed by RNA Pol II, and directed by promoter sequences at the left end of integrated LINE DNA ORF1 and ORF2 proteins bind LINE mRNA & mediate its’ nuclear transport ORF2 catalyze its reverse transcription and integration at A/T rich sites SINEs are transcribed by RNA polymerase III, and follow the same reterotransposition mechanism mediated by ORF1 & ORF2 expressed from LINE RNA from Lodishet al., Molecular Cell Biology, 5thed. Fig 10-16

7. Mutations in LINEs reduce their efficiency of transposition The vast majority of LINEs in the human genome are truncated at their 5’ end due to erroneous nature of reverse transcription The average size of LINE elements is only about 900 base pairs, whereas the full-length sequence is ≈6 kb long Only≈0.01 percent of the LINE sequences in the human genome are full-length with intact ORF1 and ORF2, ≈60–100 in total! Like LINEs, many SINEs also are truncated at their 5 end SINEs also have accumulated mutations from the time of their insertion in the germ line of an ancient ancestor of modern humans

8. NON-LTR ELEMENTS IN CANCER and TUMOR DEVELOPMENT Mobile element insertion takes place at certain characteristic sites, some corresponding to tumor suppressor genes e.g. APC (AdenomatousPolyposis Coli) L1 retroelement integration into the tumor-suppressing gene APC has been found in colon cancer patients L1 retroelement insertion into the human protooncogene c-myc was found in human breast cancinoma cells Insertions of the Alu element into the intron of the NF-1 (neurofibromatosis type I) gene may lead to neurofibromatosis Ectopic recombination between Alu elements results in chromosomal aberrations, associated with acute myeloid leukemia

9. L1 elements as cancer cell biomarkers LINE-1 methylation is identified as a molecular marker of prognosis for CM patients in stage IIIC (Sigalotti et. al, 2011) L1 elements have been hypothesized as potential molecular marker in cancer detection as L1 activity significantly increases in most human cancers (Piskareva et. al, 2011) Global hypomethylation in cells may trigger the activation of L1 element expression which may cause activation of some proto-oncogenes(Weber et. al, 2010) L1 methylation pattern can be analyzed to study the degree carcinogenesis (Aporntewan et. al.,2011)`

10. References Textbook : Molecular Cell Biology, Lodishet al., 6th ed. Research articles: • Mobile genetic elements and cancer. From mutations to gene therapy. Kozeretskaet. al. Exp Oncol 2011, 33, 4, 198–205. • Methylation levels of the “long interspersed nucleotide element-1” repetitive sequences predict survival of melanoma patients. Sigalottiet al. Journal of Translational Medicine 2011, 9:78 • The Human L1 Element: A Potential Biomarker in Cancer Prognosis, Current Status and Future Directions. Piskarevaet. al. Curr Mol Med. 2011, 11(4):286-303. • Demethylation of a LINE-1 antisense promoter in the cMet locus impairs Met signalling through induction of illegitimate transcription. Weber et. al.Oncogene 2010, 29, 5775-5784. • Hypomethylation of Intragenic LINE-1 Represses Transcription in Cancer Cells through AGO2. Aporntewanet. al.Plos One 2011, 6:3 • Disruption of the APC Gene by a Retrotransposal Insertion of LI Sequence in a Colon Cancer. Miki, Y. et. al. Cancer Reserch, 1992, 52, 643-645.