Transcription-induced chimeric RNAs, possessing sequences from different genes, are expected to increase the proteomic diversity through chimeric proteins or altered regulation. Despite their importance, few studies have focused on chimeric RNAs especially regarding their presence/roles in human cancers. By deep sequencing the transcriptome of 20 human prostate cancer and 10 matched benign prostate tissues, we obtained 1.3 billion sequence reads, which led to the identification of 2,369 chimeric RNA candidates. Chimeric RNAs occurred in significantly higher frequency in cancer than in matched benign samples. Experimental investigation of a selected 46 set led to the confirmation of 32 chimeric RNAs, of which 27 were highly recurrent and previously undescribed in prostate cancer. Importantly, a subset of these chimeras was present in prostate cancer cell lines, but not detectable in primary human prostate epithelium cells, implying their associations with cancer. These chimeras contain discernable 5′ and 3′ splice sites at the RNA junction, indicating that their formation is mediated by splicing. Their presence is also largely independent of the expression of parental genes, suggesting that other factors are involved in their production and regulation. One chimera, TMEM79-SMG5 , is highly differentially expressed in human cancer samples and therefore a potential biomarker. The prevalence of chimeric RNAs may allow the limited number of human genes to encode a substantially larger number of RNAs and proteins, forming an additional layer of cellular complexity. Together, our results suggest that chimeric RNAs are widespread, and increased chimeric RNA events could represent a unique class of molecular alteration in cancer.

Telomerase is a ribonucleoprotein (RNP) reverse transcriptase whose essential RNA subunit (TER) functions as a template for telomere repeat synthesis. Here we report the identification of two divergent TER moieties in the flowering plant Arabidopsis thaliana. Although both TER1 and TER2 copurify with telomerase activity and serve as templates for telomerase in vitro, depletion of TER1, but not TER2, leads to decreased telomerase activity and progressive telomere shortening in vivo. Moreover, mutation of the templating domain in TER1 results in the incorporation of mutant telomere repeats on chromosome ends. Thus, TER1 provides the major template for telomerase in vivo. We also show that POT1a binds TER1 with a Kd of 2 × 10 −7 M and the two components assemble into an enzymatically active RNP in vivo. In contrast, TER1-POT1b and TER2-POT1a associations were not observed. In other organisms POT1 proteins bind telomeric DNA and provide chromosome end protection. We propose that duplication of TER and POT1 in Arabidopsis fueled the evolution of novel proteinnucleic acid interactions and the migration of POT1 from the telomere to the telomerase RNP.OB fold | RNA template | gene duplication

Telomerase replenishes telomere tracts by reiteratively copying its RNA template, TER. Unlike other model organisms, Arabidopsis thaliana harbors two divergent TER genes. However, only TER1 is required for telomere maintenance. Here we examine the function of TER2. We show that TER2 is spliced and its 39 end is truncated in vivo to generate a third TER isoform, TER2 S . TERT preferentially associates with TER2 > TER1 > TER2 S . Moreover, TER2 and TER2 S assemble with Ku and POT1b (protection of telomeres), forming RNP (ribonucleoprotein) complexes distinct from TER1 RNP. Plants null for TER2 display increased telomerase enzyme activity, while TER2 overexpression inhibits telomere synthesis from TER1 and leads to telomere shortening. These findings argue that TER2 negatively regulates telomerase by sequestering TERT in a nonproductive RNP complex. Introduction of DNA double-strand breaks by zeocin leads to an immediate and specific spike in TER2 and a concomitant decrease in telomerase enzyme activity. This response is not triggered by replication stress or telomere dysfunction and is abrogated in ter2 mutants. We conclude that Arabidopsis telomerase is modulated by TER2, a novel DNA damageinduced noncoding RNA that works in concert with the canonical TER to promote genome integrity.

Dyskerin binds the H/ACA box of human telomerase RNA and is a core telomerase subunit required for RNP biogenesis and enzyme function in vivo. Missense mutations in dyskerin result in dyskeratosis congenita, a complex syndrome characterized by bone marrow failure, telomerase enzyme deficiency, and progressive telomere shortening. Here we demonstrate that dyskerin also contributes to telomere maintenance in Arabidopsis thaliana. We report that both AtNAP57, the Arabidopsis dyskerin homolog, and AtTERT, the telomerase catalytic subunit, accumulate in the plant nucleolus, and AtNAP57 associates with active telomerase RNP particles in an RNA-dependent manner. Furthermore, AtNAP57 interacts in vitro with AtPOT1a, a novel component of Arabidopsis telomerase. Although a null mutation in AtNAP57 is lethal, AtNAP57, like AtTERT, is not haploinsufficient for telomere maintenance in Arabidopsis. However, introduction of an AtNAP57 allele containing a T66A mutation decreased telomerase activity in vitro, disrupted telomere length regulation on individual chromosome ends in vivo, and established a new, shorter telomere length set point. These results imply that T66A NAP57 behaves as a dominant-negative inhibitor of telomerase. We conclude that dyskerin is a conserved component of the telomerase RNP complex in higher eukaryotes that is required for maximal enzyme activity in vivo.

Ovarian cancer is the fifth leading cause of cancer death in women. Almost 70% of ovarian cancer deaths are due to the high-grade serous subtype, which is typically detected only after it has metastasized. Characterization of high-grade serous cancer is further complicated by the significant heterogeneity and genome instability displayed by this cancer. Other than mutations in TP53, which is common to many cancers, highly recurrent recombinant events specific to this cancer have yet to be identified. Using high-throughput transcriptome sequencing of seven patient samples combined with experimental validation at DNA, RNA and protein levels, we identified a cancer-specific and inter-chromosomal fusion gene CDKN2D-WDFY2 that occurs at a frequency of 20% among sixty high-grade serous cancer samples but is absent in non-cancerous ovary and fallopian tube samples. This is the most frequent recombinant event identified so far in high-grade serous cancer implying a major cellular lineage in this highly heterogeneous cancer. In addition, the same fusion transcript was also detected in OV-90, an established high-grade serous type cell line. The genomic breakpoint was identified in intron 1 of CDKN2D and intron 2 of WDFY2 in patient tumor, providing direct evidence that this is a fusion gene. The parental gene, CDKN2D, is a cell-cycle modulator that is also involved in DNA repair, while WDFY2 is known to modulate AKT interactions with its substrates. Transfection of cloned fusion construct led to loss of wildtype CDKN2D and wildtype WDFY2 protein expression, and a gain of a short WDFY2 protein isoform that is presumably under the control of the CDKN2D promoter. The expression of short WDFY2 protein in transfected cells appears to alter the PI3K/AKT pathway that is known to play a role in oncogenesis. CDKN2D-WDFY2 fusion could be an important molecular signature for understanding and classifying sub-lineages among heterogeneous high-grade serous ovarian carcinomas.