Characterization of a Novel Exportin-1-dependent microRNA Biogenesis Pathway
The reversible state of proliferative arrest known as “cellular quiescence” plays an important role in tissue homeostasis and stem cell biology. By analyzing the expression of miRNAs and miRNA processing factors during quiescence in primary human cells, we identified a group of miRNAs that are induced during quiescence despite markedly reduced expression of Exportin-5, a protein required for canonical miRNA biogenesis. The biogenesis of these quiescence-induced miRNAs is independent of Exportin-5 and depends instead on Exportin-1. Moreover, these quiescence-induced primary miRNAs (pri-miRNAs) are modified with a 2,2,7-trimethylguanosine (TMG)-cap, which is known to help bind Exportin-1. Surprisingly, in quiescent cells Exportin-1–dependent pri-miR-34a is present in the cytoplasm together with an active small isoform of Drosha, implying the existence of a different miRNA processing pathway in these cells. Our findings suggest that during quiescence the canonical miRNA biogenesis pathway is down-regulated and specific miRNAs are generated by an alternative pathway to regulate genes involved in cellular growth arrest.
Model showing an alternative miRNA biogenesis pathway of (TMG)-capped pri-miRNAs during Cellular Quiescence (Martinez I., et al. PNAS 2017).
The Importance of Circular RNAs in the Carcinogenesis of Human Papillomavirus-related Cancers
Circular RNAs (circRNAs) represent a new type of non-coding RNA (ncRNA) that exerts important roles in carcinogenesis. Even though a handful of circRNAs were discovered more than 20 years ago (they were thought to be by-products of aberrant splicing), it was not until 2013 that several groups “re-discovered” the expression of tens of thousands of circRNAs in different organisms including humans. CircRNAs are formed by exon skipping or back-splicing events as covalently closed loops, where the 3' and 5' ends have been joined together (“head to tail” junction). CircRNAs modify gene expression by working as a sponge for microRNAs (miRNAs), being RNA binding proteins, regulating transcription, or by expressing specific proteins. Although, circRNAs have been discovered in various organisms, the molecular mechanisms by which circRNAs contribute to viral oncogenesis, such as HPV, have not been previously studied. The overall goal of this project is to identify and characterize the role of circRNAs in the process of carcinogenesis in HPV-related cancers such as cervical and head and neck cancers.
Potential regulation of human circular RNAs (circRNAs) by high-risk Human Papillomavirus (HPV) oncogenes E6 and E7 during the process of carcinogenesis.
The Role of Long Non-coding RNAs in the Carcinogenesis of Human Papillomavirus-related Cancers
In 2009, John Rinn and Eric Lander’s labs at the Broad Institute identified over a thousand long non-coding RNAs (lncRNAs) that are highly conserved in mammals. To date, there are more than 145,000 human annotated lncRNAs identified. LncRNAs are RNAs larger than 200 nucleotides long that do not encode for a protein and they can function by acting as signal, guide, decoy, or scaffold RNAs. Increasing evidence suggests that lncRNAs are main players in the process of carcinogenesis and they have been involved in most of the hallmarks of human cancer progression. Recently, our laboratory discovered several human lncRNAs differentially expressed by the presence of HPV-16 E6 including GAS5, H19, and FAM83H-AS1. Interestingly, FAM83H-AS1 was found overexpressed in HPV-16 positive cervical cancer cell lines in an HPV-16 E6-dependent manner but independently of p53 regulation. Furthermore, FAM83H-AS1 was found to be regulated through the E6-p300 pathway. Knockdown of FAM83H-AS1 by siRNAs decreased cellular proliferation, migration and increased apoptosis. FAM83H-AS1 was also found to be altered in human cervical cancer tissues and high expression of this lncRNA was associated with worse overall survival, suggesting an important role in cervical carcinogenesis. The goal of this project is to elucidate the importance of specific lncRNAs in HPV-related cancers, so we can provide a framework whereby new evidence-based therapies can be developed.
Increased FAM83H-AS1 expression in primary cervical keratinocytes (HCK) containing the HPV-16 genome as well as in HPV-16 positive cervical cancer and head and neck squamous cell carcinoma (HNSCC) cell lines. FAM83H-AS1 knockdown altered cell migration and high FAM83H-AS1 expression correlates with poor overall survival.
The Significance of Long Non-coding RNAs in Cellular Radiation Therapy Response in Non-small Cell Lung Cancers
Worldwide, lung cancer is the leading cause of death by cancer in both men and women, with a mortality rate higher than prostate, breast, and colon cancer combined. Even though the average overall 5-year survival rate is about 17.8%, women have an overall survival rate two times greater than men. Studies have shown that women tend to respond better to chemotherapy and radiation, but explanations regarding the molecular mechanisms behind these differences in response rates are lacking. Long non-coding RNAs (lncRNAs) are >200 nucleotides long, usually lack an open reading frame, and function mainly as genetic regulators at both the transcriptional and post-transcriptional level. Dysregulation of lncRNAs has been shown to attribute to cancer formation and resistance to chemotherapy and radiation therapy. The objective of this project is to investigate the potential role that lncRNAs may have in the difference between men and women in their response rate to radiation therapy in Non-small Cell Lung Cancers (NSCLC).
Differences in radiation sensitivity in several non-small cell lung cancer (NSCLC) cell lines.