Biology subjects

Daugaard, I.

Publications and source records attributed to Daugaard, I..

5 recordsLinked to original sources

bioRxiv2018

Non-coding AUG circRNAs constitute an abundant and conserved subclass of circles

Circular RNAs (circRNAs) are a subset of noncoding RNAs (ncRNAs) previously considered as products of missplicing. Now, circRNAs are considered functional molecules, although to date, only few functions have been experimentally validated, and therefore the vast majority of circRNAs are without known relevance. Here, based on RNA sequencing from the ENCODE consortium, we identify and characterize a subset of circRNAs, coined AUG circRNAs, defined by spanning the canonical translational start site in the protein-coding host genes. AUG circRNAs are more abundantly expressed and conserved than other groups of circRNAs, and they display an Alu-independent mechanism of biogenesis. The AUG circRNAs contain part of bona fide ORF, and in the recent years, several studies have reported cases of circRNA translation. However, using thorough cross-species analysis, extensive ribosome profiling analyses and experimental data on a selected panel of AUG circRNAs, we observe no indications of translation of AUG circRNAs or any other circRNAs. Our data provide a comprehensive classification of circRNAs and, collectively, the analyses suggest that the AUG circRNAs constitute an abundant subclass of circRNAs produced independently of primate-specific Alu elements. Moreover, AUG circRNAs exhibit high cross-species conservation and are therefore likely to be functionally relevant.

molecular biology↗

bioRxiv2017

miR-128 inhibits telomerase activity by targeting TERT mRNA

Telomerase is a unique cellular reverse transcriptase essential for maintaining telomere stability and required for the unlimited proliferation of cancer cells. The limiting determinant of telomerase activity is the catalytic component TERT, and TERT expression is closely correlated with telomerase activity and cancer initiation and disease progression. For this reason the regulation of TERT levels in the cell is of great importance. microRNAs (miRs) function as an additional regulatory level in cells, crucial for defining expression boundaries, proper cell fate decisions, cell cycle control, genome integrity, cell death and metastasis. We performed an anti-miR library screen to identity novel miRs, which participate in the control of telomerase. We identified the tumor suppressor miR (miR-128) as a novel endogenous telomerase inhibitor and determined that miR-128 significantly reduces the mRNA and protein levels of Tert in a panel of cancer cell lines. We further evaluated the mechanism by which miR-128 regulates TERT and demonstrated that miR-128 interacts directly with the coding sequence of TERT mRNA in both HeLa cells and teratoma cells. Interestingly, the functional miR-128 binding site in TERT mRNA, is conserved between TERT and the other cellular reverse transcriptase encoded by Long Interspaced Elements-1 (LINE-1 or L1), which can also contribute to the oncogenic phenotype of cancer. This finding supports the novel idea that miRs may function in parallel pathways to inhibit tumorigenesis, by regulating a group of enzymes (RT) by targeting conserved binding sites in the coding region of both enzymes.\n\nNOVELTY AND IMPACTTelomerase is an RNA-dependent DNA polymerase that synthesizes telomeric DNA sequences and almost universally provides the molecular basis for unlimited proliferative potential. Expression of human telomerase alone is sufficient for the immortalization of diverse cell types. We have identified the tumor suppressor microRNA (miR-128) as a novel regulator of telomerase, which directly targets the coding sequence (CDS) of TERT mRNA and significantly represses Tert protein expression in a panel of cancer cell lines.

cancer biology↗

bioRxiv2017

miR-128-induced LINE-1 restriction is dependent on down-regulation of hnRNPA1

The majority of the human genome is made of transposable elements, giving rise to interspaced repeats, including Long Interspersed Element-1s (LINE-1s or L1s). L1s are active human DNA parasites involved in genomic diversity and evolution, but can also contribute to genomic instability and diseases. L1s require host factors to complete their life cycles, whereas the host has evolved numerous mechanisms to restrict L1-induced mutagenesis. Restriction mechanisms in somatic cells include methylation of the L1 promoter, anti-viral factors and RNA-mediated processes such as small RNAs. microRNAs (miRNAs or miRs) are small non-coding RNAs that post-transcriptionally repress multiple target genes often found in the same cellular pathways. We have recently established that the interferon-inducible miR-128 function as a novel restriction factor inhibiting L1 mobilization in somatic cells. We have further demonstrated that miR-128 function through a dual mechanism; by directly targeting L1 RNA for degradation and indirectly by inhibiting a cellular co-factor which L1 is dependent on to transpose to new genomic locations (TNPO1). Here we add another piece to the puzzle of the enigmatic L1 life cycle. We show that miR-128 also inhibits another key cellular factor, hnRNPA1, by significantly reducing mRNA and protein levels through direct interaction with the coding sequence (CDS) of hnRNPA1 mRNA. Furthermore, we demonstrate that repression of hnRNPA1 using shRNA significantly decreases de novo L1 retrotransposition and that induced hnRNPA1 expression enhances L1 mobilization. Finally, we determine that hnRNPA1 is a functional target of miR-128 and that induced hnRNPA1 expression in miR-128-overexpressing cells can partly rescue the miR-128-induced repression of L1s ability to transpose to different genomic locations. Thus, we have identified an additional mechanism by which miR-128 represses L1 retrotransposition and mediate genomic stability.

cell biology↗

bioRxiv2017

MicroRNA miR-128 represses LINE-1 (L1) retrotransposition by downregulating the nuclear import factor TNPO1

Repetitive elements, including LINE-1 (L1), comprise approximately half of the human genome. These elements can potentially destabilize the genome by initiating their own replication and reintegration into new sites (retrotransposition). In somatic cells, transcription of L1 elements are repressed by distinct molecular mechanisms including DNA methylation and histone modifications to repress transcription. Under conditions of hypomethylation (e.g. in tumor cells) a window of opportunity for L1 de-repression arises and additional restriction mechanisms become crucial. We recently demonstrated that the microRNA miR-128 represses L1 activity by directly binding to L1 ORF2 RNA. In this study, we tested whether miR-128 can also control L1 activity by repressing cellular proteins important for L1 retrotransposition. We found that miR-128 targets the 3UTR of the nuclear import factor transportin 1 (TNPO1) mRNA. Manipulation of miR-128 and TNPO1 levels demonstrated that induction or depletion of TNPO1 affects L1 retrotransposition and nuclear import of an L1-RNP complex (using L1-encoded ORF1p as a proxy for L1-RNP complexes). Moreover, TNPO1 overexpression partially reversed the repressive effect of miR-128 on L1 retrotransposition. Our study represents the first description of a protein factor involved in nuclear import of the L1 element and demonstrates that miR-128 controls L1 activity in somatic cells through two independent mechanisms: direct binding to L1 RNA, and regulating a cellular factor necessary for L1 nuclear import and retrotransposition.

cell biology↗

bioRxiv2017

Interferon-inducible miR-128 modulates HIV-1 replication by targeting TNPO3 mRNA

The HIV/AIDS pandemic remains an important threat to human health. We have recently demonstrated that a novel microRNA (miR-128) represses retrotransposon (LINE-1 or L1) by a dual mechanism, by directly targeting the coding region of the L1 RNA and by repressing a required nuclear import factor (TNPO1). We have further determined that miR-128 represses the expression of all three isoforms of TNPO proteins (transportins, TNPO1,-2 and TNPO3). Here, we establish that miR-128 also controls HIV-1 replication by repressing TNPO3. TNPO3 is well established to regulate HIV-1 nuclear import and viral replication. Here, we report that the type I interferon inducible miR-128 directly targets two sites in the TNPO3 mRNA, significantly down-regulating TNPO3 mRNA and protein expression levels. Manipulation of miR-128 levels in HIV target cell lines and in primary human CD4 T-cells by over-expression or knockdown showed that modulation of TNPO3 by miR-128 affects HIV-1 replication but not MLV infection. In addition, we found that miR-128 modulation of HIV-1 replication is reduced with TNPO3-independent HIV-1 virus and in cells depleted of CPSF6, suggesting that miR-128-indued TNPO3 repression is partly required for miR-128-induced inhibition of HIV-1 replication. Finally, challenging miR-modulated Jurkat cells or primary CD4 T-cells with wildtype, replication-competent HIV-1 shows that miR-128 significantly delays spreading infection. Thus, we have established a novel role of miR-128 in anti-viral defense in human cells, inhibiting HIV-1 replication partly by targeting TNPO3.

cell biology↗