Using a modified RNA-sequencing (RNA-seq) approach, we discovered a new family of unusually short RNAs mapping to ribosomal RNA 5.8S, which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. Using a new quantitative detection method that we developed, we confirmed our RNA-seq data and determined that the minimal core doRNA sequence and its 13-nt variant C-doRNA (doRNA with a 5′ Cytosine) are the two most abundant doRNAs, which, together, may outnumber microRNAs. The C-doRNA/doRNA ratio is stable within species but differed between species. doRNA and C-doRNA are mainly cytoplasmic and interact with heterogeneous nuclear ribonucleoproteins (hnRNP) A0, A1 and A2B1, but not Argonaute 2. Reporter gene activity assays suggest that C-doRNA may function as a regulator of Annexin II receptor (AXIIR) expression. doRNAs are differentially expressed in prostate cancer cells/tissues and may control cell migration. These findings suggest that unusually short RNAs may be more abundant and important than previously thought.

Keratinocyte carcinomas are among the most prevalent malignancies worldwide. Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC) are the two cancers recognized as keratinocyte carcinomas. The standard of care for treating these cancers includes surgery and ablative therapies. However, in recent years, targeted therapies (e.g., cetuximab for cSCC and vismodegib/sonidegib for BCC) have been used to treat advanced disease as well as immunotherapy (e.g., cemiplimab). These treatments are expensive and have significant toxicities with objective response rates approaching ~50–65%. Hence, there is a need to dissect the molecular pathogenesis of these cancers to identify novel biomarkers and therapeutic targets to improve disease management. Several cancer-testis antigens (CTA) and developmental genes (including embryonic stem cell factors and fetal genes) are ectopically expressed in BCC and cSCC. When ectopically expressed in malignant tissues, functions of these genes may be recaptured to promote tumorigenesis. CTAs and developmental genes are emerging as important players in the pathogenesis of BCC and cSCC, positioning themselves as attractive candidate biomarkers and therapeutic targets requiring rigorous testing. Herein, we review the current research and offer perspectives on the contributions of CTAs and developmental genes to the pathogenesis of keratinocyte carcinomas.

Retrotransposons have played an important role in evolution through their transposable activity. The largest and the only currently active human group of mobile DNAs are the LINE-1 retrotransposons. The ectopic expression of LINE-1 has been correlated with genomic instability. Narrow-band ultraviolet B (NB-UVB) and broad-band ultraviolet B (BB-UVB) phototherapy is commonly used for the treatment of dermatological diseases. UVB exposure is carcinogenic and can lead, in keratinocytes, to genomic instability. We hypothesize that LINE-1 reactivation occurs at a high rate in response to UVB exposure on the skin, which significantly contributes to genomic instability and DNA damage leading to cellular senescence and photoaging. Immortalized N/TERT1 and HaCaT human keratinocyte cell lines were irradiated in vitro with either NB-UVB or BB-UVB. Using immunofluorescence and Western blotting, we confirmed UVB-induced protein expression of LINE-1. Using RT-qPCR, we measured the mRNA expression of LINE-1 and senescence markers that were upregulated after several NB-UVB exposures. Selected miRNAs that are known to bind LINE-1 mRNA were measured using RT-qPCR, and the expression of miR-16 was downregulated with UVB exposure. Our findings demonstrate that UVB irradiation induces LINE-1 reactivation and DNA damage in normal keratinocytes along with the associated upregulation of cellular senescence markers and change in miR-16 expression.

Small RNA sequencing (sRNA-Seq) approaches unveiled sequences derived from longer non-coding RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA) fragments, known as tRFs and rRFs, respectively. However, rRNAs and RNAs shorter than 16 nt are often depleted from library preparations/sequencing analyses, although they may be functional. Here, we sought to obtain a complete repertoire of small RNAs by sequencing the total RNA from 11 samples of 6 different eukaryotic organisms, from yeasts to human, in an extended 8- to 30-nt window of RNA length. The 8- to 15-nt window essentially contained fragments of longer non-coding RNAs, such as microRNAs, PIWI-associated RNAs (piRNAs), small nucleolar RNAs (snoRNAs), tRNAs and rRNAs. Notably, unusually short RNAs < 16 nt were more abundant than those >16 nt in bilaterian organisms. A new RT-qPCR method confirmed that two unusually short rRFs of 12 and 13 nt were more overly abundant (~3-log difference) than two microRNAs. We propose to not deplete rRNA and to reduce the lower threshold of RNA length to include unusually short RNAs in sRNA-Seq analyses and datasets, as their abundance and diversity support their potential role and importance as biomarkers of disease and/or mediators of cellular function.

Genomic instability is a prominent hallmark of cancer, however the mechanisms that drive and sustain this process remain elusive. Research demonstrates that numerous cancers with increased levels of genomic instability ectopically express meiosis-specific genes and undergo meiomitosis, the clash of mitotic and meiotic processes. These meiotic genes may represent novel therapeutic targets for the treatment of cancer. We studied the relationship between the expression of the meiosis protein HORMAD1 and genomic instability in squamous cell carcinomas (SCCs). First, we assessed markers of DNA damage and genomic instability following knockdown and overexpression of HORMAD1 in different cell lines representing SCCs and epithelial cancers. shRNA-mediated depletion of HORMAD1 expression resulted in increased genomic instability, DNA damage, increased sensitivity to etoposide, and decreased expression of DNA damage response/repair genes. Conversely, overexpression of HORMAD1 exhibited protective effects leading to decreased DNA damage, enhanced survival and decreased sensitivity to etoposide. Furthermore, we identified a meiotic molecular pathway that regulates HORMAD1 expression by targeting the upstream meiosis transcription factor STRA8. Our results highlight a specific relationship between HORMAD1 and genomic instability in SCCs, suggesting that selectively inhibiting HORMAD1, possibly, through STRA8 signaling, may provide a new paradigm of treatment options for HORMAD1-expressing SCCs.

Recently, we discovered a new family of unusually short RNAs mapping to 5.8S ribosomal RNA (rRNA) and which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. To confirm these small RNA-sequencing (RNA-Seq) data, validate the existence of the two overly abundant doRNAs—the minimal core 12-nt doRNA sequence and its + 1-nt variant bearing a 5′ Cytosine, C-doRNA—and streamline their analysis, we developed a new specific and sensitive splinted 5′ ligation reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) method. This method is based on a splint-assisted ligation of an adapter to the 5′ end of doRNAs, followed by RT-qPCR amplification and quantitation. Our optimized protocol, which may discriminate between doRNA, C-doRNA, mutated and precursor sequences, can accurately detect as low as 240 copies and is quantitatively linear over a range of 7 logs. This method provides a unique tool to expand and facilitate studies exploring the molecular and cellular biology of RNA species shorter than microRNAs.