Genetic Variants Associated with Long-Terminal Repeats Can Diagnostically Classify Cannabis Varieties.

Cannabis sativa (Cannabis) has recently been legalized in multiple countries globally for either its recreational or medicinal use. This, in turn, has led to a marked increase in the number of Cannabis varieties available for use in either market. However, little information currently exists on the genetic distinction between adopted varieties. Such fundamental knowledge is of considerable value and underpins the accelerated development of both a nascent pharmaceutical industry and the commercial recreational market. Therefore, in this study, we sought to assess genetic diversity across 10 Cannabis varieties by undertaking a reduced representation shotgun sequencing approach on 83 individual plants to identify variations which could be used to resolve the genetic structure of the assessed population. Such an approach also allowed for the identification of the genetic features putatively associated with the production of secondary metabolites in Cannabis. Initial analysis identified 3608 variants across the assessed population with phylogenetic analysis of this data subsequently enabling the confident grouping of each variety into distinct subpopulations. Within our dataset, the most diagnostically informative single nucleotide polymorphisms (SNPs) were determined to be associated with the long-terminal repeat (LTRs) class of retroelements, with 172 such SNPs used to fully resolve the genetic structure of the assessed population. These 172 SNPs could be used to design a targeted resequencing panel, which we propose could be used to rapidly screen different Cannabis plants to determine genetic relationships, as well as to provide a more robust, scientific classification of Cannabis varieties as the field moves into the pharmaceutical sphere.

Regulated post-transcriptional RNA cleavage diversifies the eukaryotic transcriptome.

The complexity of the eukaryotic transcriptome is generated by the interplay of transcription initiation, termination, alternative splicing, and other forms of post-transcriptional modification. It was recently shown that RNA transcripts may also undergo cleavage and secondary 5' capping. Here, we show that post-transcriptional cleavage of RNA contributes to the diversification of the transcriptome by generating a range of small RNAs and long coding and noncoding RNAs. Using genome-wide histone modification and RNA polymerase II occupancy data, we confirm that the vast majority of intraexonic CAGE tags are derived from post-transcriptional processing. By comparing exonic CAGE tags to tissue-matched PARE data, we show that the cleavage and subsequent secondary capping is regulated in a developmental-stage- and tissue-specific manner. Furthermore, we find evidence of prevalent RNA cleavage in numerous transcriptomic data sets, including SAGE, cDNA, small RNA libraries, and deep-sequenced size-fractionated pools of RNA. These cleavage products include mRNA variants that retain the potential to be translated into shortened functional protein isoforms. We conclude that post-transcriptional RNA cleavage is a key mechanism that expands the functional repertoire and scope for regulatory control of the eukaryotic transcriptome.

MicroRNAs-140-5p/140-3p modulate Leydig cell numbers in the developing mouse testis.

MicroRNAs (miRNAs) have been shown to play key regulatory roles in a range of biological processes, including cell differentiation and development. To identify miRNAs that participate in gonad differentiation, a fundamental and tightly regulated developmental process, we examined miRNA expression profiles at the time of sex determination and during the early fetal differentiation of mouse testes and ovaries using high-throughput sequencing. We identified several miRNAs that were expressed in a sexually dimorphic pattern, including several members of the let-7 family, miR-378, and miR-140-3p. We focused our analysis on the most highly expressed, sexually dimorphic miRNA, miR-140-3p, and found that both miR-140-3p and its more lowly expressed counterpart, the previously annotated guide strand, miR-140-5p, are testis enriched and expressed in testis cords. Analysis of the miR-140-5p/miR-140-3p-null mouse revealed a significant increase in the number of Leydig cells in the developing XY gonad, strongly suggesting an important role for miR-140-5p/miR-140-3p in testis differentiation in mouse.

Expression of distinct RNAs from 3' untranslated regions.

The 3' untranslated regions (3'UTRs) of eukaryotic genes regulate mRNA stability, localization and translation. Here, we present evidence that large numbers of 3'UTRs in human, mouse and fly are also expressed separately from the associated protein-coding sequences to which they are normally linked, likely by post-transcriptional cleavage. Analysis of CAGE (capped analysis of gene expression), SAGE (serial analysis of gene expression) and cDNA libraries, as well as microarray expression profiles, demonstrate that the independent expression of 3'UTRs is a regulated and conserved genome-wide phenomenon. We characterize the expression of several 3'UTR-derived RNAs (uaRNAs) in detail in mouse embryos, showing by in situ hybridization that these transcripts are expressed in a cell- and subcellular-specific manner. Our results suggest that 3'UTR sequences can function not only in cis to regulate protein expression, but also intrinsically and independently in trans, likely as noncoding RNAs, a conclusion supported by a number of previous genetic studies. Our findings suggest novel functions for 3'UTRs, as well as caution in the use of 3'UTR sequence probes to analyze gene expression.

Identification of novel non-coding RNAs using profiles of short sequence reads from next generation sequencing data.

BACKGROUND: The increasing interest in small non-coding RNAs (ncRNAs) such as microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs) and recent advances in sequencing technology have yielded large numbers of short (18-32 nt) RNA sequences from different organisms, some of which are derived from small nucleolar RNAs (snoRNAs) and transfer RNAs (tRNAs). We observed that these short ncRNAs frequently cover the entire length of annotated snoRNAs or tRNAs, which suggests that other loci specifying similar ncRNAs can be identified by clusters of short RNA sequences. RESULTS: We combined publicly available datasets of tens of millions of short RNA sequence tags from Drosophila melanogaster, and mapped them to the Drosophila genome. Approximately 6 million perfectly mapping sequence tags were then assembled into 521,302 tag-contigs (TCs) based on tag overlap. Most transposon-derived sequences, exons and annotated miRNAs, tRNAs and snoRNAs are detected by TCs, which show distinct patterns of length and tag-depth for different categories. The typical length and tag-depth of snoRNA-derived TCs was used to predict 7 previously unrecognized box H/ACA and 26 box C/D snoRNA candidates. We also identified one snRNA candidate and 86 loci with a high number of tags that are yet to be annotated, 7 of which have a particular 18mer motif and are located in introns of genes involved in development. A subset of new snoRNA candidates and putative ncRNA candidates was verified by Northern blot. CONCLUSIONS: In this study, we have introduced a new approach to identify new members of known classes of ncRNAs based on the features of TCs corresponding to known ncRNAs. A large number of the identified TCs are yet to be examined experimentally suggesting that many more novel ncRNAs remain to be discovered.

Germline polymorphisms in an enhancer of PSIP1 are associated with progression-free survival in epithelial ovarian cancer.

Women with epithelial ovarian cancer (EOC) are usually treated with platinum/taxane therapy after cytoreductive surgery but there is considerable inter-individual variation in response. To identify germline single-nucleotide polymorphisms (SNPs) that contribute to variations in individual responses to chemotherapy, we carried out a multi-phase genome-wide association study (GWAS) in 1,244 women diagnosed with serous EOC who were treated with the same first-line chemotherapy, carboplatin and paclitaxel. We identified two SNPs (rs7874043 and rs72700653) in TTC39B (best P=7x10-5, HR=1.90, for rs7874043) associated with progression-free survival (PFS). Functional analyses show that both SNPs lie in a putative regulatory element (PRE) that physically interacts with the promoters of PSIP1, CCDC171 and an alternative promoter of TTC39B. The C allele of rs7874043 is associated with poor PFS and showed increased binding of the Sp1 transcription factor, which is critical for chromatin interactions with PSIP1. Silencing of PSIP1 significantly impaired DNA damage-induced Rad51 nuclear foci and reduced cell viability in ovarian cancer lines. PSIP1 (PC4 and SFRS1 Interacting Protein 1) is known to protect cells from stress-induced apoptosis, and high expression is associated with poor PFS in EOC patients. We therefore suggest that the minor allele of rs7874043 confers poor PFS by increasing PSIP1 expression.

Methylome sequencing in triple-negative breast cancer reveals distinct methylation clusters with prognostic value.

Epigenetic alterations in the cancer methylome are common in breast cancer and provide novel options for tumour stratification. Here, we perform whole-genome methylation capture sequencing on small amounts of DNA isolated from formalin-fixed, paraffin-embedded tissue from triple-negative breast cancer (TNBC) and matched normal samples. We identify differentially methylated regions (DMRs) enriched with promoters associated with transcription factor binding sites and DNA hypersensitive sites. Importantly, we stratify TNBCs into three distinct methylation clusters associated with better or worse prognosis and identify 17 DMRs that show a strong association with overall survival, including DMRs located in the Wilms tumour 1 (WT1) gene, bi-directional-promoter and antisense WT1-AS. Our data reveal that coordinated hypermethylation can occur in oestrogen receptor-negative disease, and that characterizing the epigenetic framework provides a potential signature to stratify TNBCs. Together, our findings demonstrate the feasibility of profiling the cancer methylome with limited archival tissue to identify regulatory regions associated with cancer.

Nuclear-localized tiny RNAs are associated with transcription initiation and splice sites in metazoans.

We have recently shown that transcription initiation RNAs (tiRNAs) are derived from sequences immediately downstream of transcription start sites. Here, using cytoplasmic and nuclear small RNA high-throughput sequencing datasets, we report the identification of a second class of nuclear-specific approximately 17- to 18-nucleotide small RNAs whose 3' ends map precisely to the splice donor site of internal exons in animals. These splice-site RNAs (spliRNAs) are associated with highly expressed genes and show evidence of developmental stage- and region-specific expression. We also show that tiRNAs are localized to the nucleus, are enriched at chromatin marks associated with transcription initiation and possess a 3'-nucleotide bias. Additionally, we find that microRNA-offset RNAs (moRNAs), the miR-15/16 cluster previously linked to oncosuppression and most small nucleolar RNA (snoRNA)-derived small RNAs (sdRNAs) are enriched in the nucleus, whereas most miRNAs and two H/ACA sdRNAs are cytoplasmically enriched. We propose that nuclear-localized tiny RNAs are involved in the epigenetic regulation of gene expression.

Touchdown PCR for increased specificity and sensitivity in PCR amplification.

Touchdown (TD) PCR offers a simple and rapid means to optimize PCRs, increasing specificity, sensitivity and yield, without the need for lengthy optimizations and/or the redesigning of primers. TD-PCR employs an initial annealing temperature above the projected melting temperature (T(m)) of the primers being used, then progressively transitions to a lower, more permissive annealing temperature over the course of successive cycles. Any difference in T(m) between correct and incorrect annealing will produce an exponential advantage of twofold per cycle. TD-PCR has found wide applicability in standard PCR protocols, including reverse transcriptase-dependent PCR, as well as in the generation of cDNA libraries and single nucleotide polymorphism screening. TD-PCR is particularly useful for templates that are difficult to amplify but can also be standardly used to enhance specificity and product formation. The procedure takes between 90 and 120 min, depending on the template length.

Molecular evolution of the HBII-52 snoRNA cluster.

HBII-52 is a human brain-specific C/D box snoRNA that potentially regulates the editing and/or alternative splicing of the serotonin receptor. Forty-two nearly identical copies of the HBII-52 gene are located immediately downstream of the SNRPN protein-coding gene in an imprinted locus associated with Prader-Willi syndrome. Other eutherian mammals, with genomic assemblies covering the corresponding locus, also have multiple orthologous copies of HBII-52. The SNRPB gene, which is known to have given rise to SNRPN through gene duplication, expresses a C/D box snoRNA, SNORD119, from its fifth intron. Here we show that, despite the fact that they lie in different positions relative to the orthologous SNRPB/SNRPN coding sequences, there are significant sequence similarities between SNORD119 and HBII-52, including the antisense element and the stem-forming regions. By analysing these snoRNAs in marsupial and eutherian mammal genomes, we reconstruct the likely evolutionary history of the HBII-52 cluster and SNORD119 and suggest that they have evolved from a common ancestor.