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-LTR Retrotransposons 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  Alu elements 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 Lodish et al., Molecular Cell Biology, 5 th ed. 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 Lodish et al., Molecular Cell Biology, 5 th ed. 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 (Adenomatous Polyposis 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, Lodish et al., 6 th ed.  Research articles: 1. Mobile genetic elements and cancer. From mutations to gene therapy. Kozeretska et. al. Exp Oncol 2011, 33, 4, 198–205. 2. Methylation levels of the “long interspersed nucleotide element-1” repetitive sequences predict survival of melanoma patients. Sigalotti et al. Journal of Translational Medicine 2011, 9:78 3. The Human L1 Element: A Potential Biomarker in Cancer Prognosis, Current Status and Future Directions. Piskareva et. al. Curr Mol Med. 2011, 11(4):286-303. 4. 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. 5. Hypomethylation of Intragenic LINE-1 Represses Transcription in Cancer Cells through AGO2. Aporntewan et. al. Plos One 2011, 6:3 6. 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.