miR-128 could potentially regulate telomerase activity by directly targeting TERT mRNA, or by regulating expression of other proteins that regulate telomerase, or both. In parallel with this work our laboratory have been investigating the mechanisms by which miR-128 regulate L1-induced mutagenesis, including the repression of L1 RT activity. We have demonstrated that miR-128 repress L1 retrotransposition and genomic integration by directly targeting the L1 RNA, in a similar fashion as when miRs function as an anti-viral defense mechanism in human cells, limiting viral replication of RNA virus [29, 38, 39]. We surprisingly determined that miR-128 targets L1 RNA by binding directly through an imperfect seed match to L1 RNA in the coding sequence (CDS) of ORF2, which encodes the L1 endonuclease and reverse transcriptase (RT). With our new finding that miR-128 can also repress telomerase activity in a functional assay (q-TRAP) and by regulating TERT mRNA and protein levels (Figure 2), we hypothesized that miR-128 might be binding to a shared conserved site between L1 RT and TERT mRNA. We aligned the mRNA sequences of the two cellular RTs (L1 and TERT) and determined that the functional miR-128 binding site in L1 is, in fact, present at two locations in the CDS of TERT mRNA (Figure 3A and 3B). To test if the two non-canonical miR-128 seed sites are functional, we generated TERT CDS luciferase constructs either encoding the wildtype (WT) TERT binding Site #1 or binding Site #2. In addition, we generated a 23nt perfect miR-128 match positive control plasmid (as previously described [29, 40]). HeLa cells were co-transfected with one of the three TERT constructs WT Site #1, WT Site #2 or the positive control, (Wildtype (WT) seed plasmids) and either miR-128 or miR control mimic oligonucleotides. Luciferase activity was modestly, but significantly reduced in HeLa cells transfected with either one of the two WT TERT constructs and miR-128, relative to miR controls (Figure 3C). As expected, luciferase activity was potently repressed in the positive HeLa cell control (Figure 3C). These experiments supports the conclusion that miR-128 can bind to the predicted binding site likely at both location of the TERT mRNA sequence. Next, we generated a plasmid encoding the TERT binding sites (Site #2) in which we included mutations in the putative miR-128–binding site of TERT mRNA (Figure 3D, top). HeLa cells co-transfected with the plasmid encoding WT TERT (WT seed plasmid) and miR-128, showed a significant reduced luciferase activity as previously demonstrated (Figure 3C and 3D). In contrast, HeLa cells co-transfected with the mutant TERT mRNA-binding site (Mutated seed plasmid) and either mature miR-128 or control-miR mimics exhibited luciferase activity at similar levels as in the WT TERT and control-miR cells, consistent with miR-128 no longer binding and repressing luciferase reporter-gene expression (Figure 3D). These experiments determine that miR-128 is dependent on the predicted nucleotide sequence to interact with TERT mRNA.