Functional filter for whole-genome sequencing data identifies HHT and stress-associated non-coding SMAD4 polyadenylation site variants >5 kb from coding DNA.

Despite whole-genome sequencing (WGS), many cases of single-gene disorders remain unsolved, impeding diagnosis and preventative care for people whose disease-causing variants escape detection. Since early WGS data analytic steps prioritize protein-coding sequences, to simultaneously prioritize variants in non-coding regions rich in transcribed and critical regulatory sequences, we developed GROFFFY, an analytic tool that integrates coordinates for regions with experimental evidence of functionality. Applied to WGS data from solved and unsolved hereditary hemorrhagic telangiectasia (HHT) recruits to the 100,000 Genomes Project, GROFFFY-based filtration reduced the mean number of variants/DNA from 4,867,167 to 21,486, without deleting disease-causal variants. In three unsolved cases (two related), GROFFFY identified ultra-rare deletions within the 3' untranslated region (UTR) of the tumor suppressor SMAD4, where germline loss-of-function alleles cause combined HHT and colonic polyposis (MIM: 175050). Sited >5.4 kb distal to coding DNA, the deletions did not modify or generate microRNA binding sites, but instead disrupted the sequence context of the final cleavage and polyadenylation site necessary for protein production: By iFoldRNA, an AAUAAA-adjacent 16-nucleotide deletion brought the cleavage site into inaccessible neighboring secondary structures, while a 4-nucleotide deletion unfolded the downstream RNA polymerase II roadblock. SMAD4 RNA expression differed to control-derived RNA from resting and cycloheximide-stressed peripheral blood mononuclear cells. Patterns predicted the mutational site for an unrelated HHT/polyposis-affected individual, where a complex insertion was subsequently identified. In conclusion, we describe a functional rare variant type that impacts regulatory systems based on RNA polyadenylation. Extension of coding sequence-focused gene panels is required to capture these variants.

A Global Survey of the Full-Length Transcriptome of Apis mellifera by Single-Molecule Long-Read Sequencing.

As important pollinators, honey bees play a crucial role in both maintaining the ecological balance and providing products for humans. Although several versions of the western honey bee genome have already been published, its transcriptome information still needs to be refined. In this study, PacBio single-molecule sequencing technology was used to sequence the full-length transcriptome of mixed samples from many developmental time points and tissues of A. mellifera queens, workers and drones. A total of 116,535 transcripts corresponding to 30,045 genes were obtained. Of these, 92,477 transcripts were annotated. Compared to the annotated genes and transcripts on the reference genome, 18,915 gene loci and 96,176 transcripts were newly identified. From these transcripts, 136,554 alternative splicing (AS) events, 23,376 alternative polyadenylation (APA) sites and 21,813 lncRNAs were detected. In addition, based on the full-length transcripts, we identified many differentially expressed transcripts (DETs) between queen, worker and drone. Our results provide a complete set of reference transcripts for A. mellifera that dramatically expand our understanding of the complexity and diversity of the honey bee transcriptome.

CPSF6-mediated XBP1 3'UTR shortening attenuates cisplatin-induced ER stress and elevates chemo-resistance in lung adenocarcinoma.

Alternative polyadenylation (APA) is a widespread mechanism generating RNA molecules with alternative 3' ends. Herein, we discovered that TargetScan includes a novel XBP1 transcript with a longer 3' untranslated region (UTR) (XBP1-UL) than that included in NCBI. XBP1-UL exhibited a lowered level in blood samples from lung adenocarcinoma (LUAD) patients and in those after DDP treatment. Consistently, XBP1-UL was reduced in A549 cells compared to normal BEAS-2B cells, as well as in DDP-treated/resistant A549 cells relative to controls. Moreover, due to decreased usage of the distal polyadenylation site (PAS) in 3'UTR, XBP1-UL level was lowered in A549 cells and decreased further in DDP-resistant A549 (A549/DDP) cells. Importantly, use of the distal PAS (dPAS) and XBP1-UL level were gradually reduced in A549 cells under increasing concentrations of DDP, which was attributed to DDP-induced endoplasmic reticulum (ER) stress. Furthermore, XBP1 transcripts with shorter 3'UTR (XBP1-US) were more stable and presented stronger potentiation on DDP resistance. The choice of proximal PAS (pPAS) was attributed to CPSF6 elevation, which was caused by BRCA1-distrupted R-loop accumulation in CPSF6 5'end. DDP-induced nuclear LINC00221 also facilitated CPSF6-induced pPAS choice in the pre-XBP1 3'end. Finally, we found that unlike the unspliced XBP1 protein (XBP1-u), the spliced form XBP1-s retarded p53 degradation to facilitate DNA damage repair of LUAD cells. The current study provides new insights into tumor progression and DDP resistance in LUAD, which may contribute to improved LUAD treatment.

Polymorphism in the human arylamine N-acetyltransferase 1 gene 3'-untranslated region determines polyadenylation signal usage.

Human arylamine N-acetyltransferase 1 (NAT1) encodes a drug-metabolising enzyme that plays a role in chemical-associated cancer risk, cancer cell survival and mitochondrial function. Its expression and protein activity are regulated by transcriptional, translational, and post-translational processes, including microRNAs such as miR-1290. Several studies have shown the presence of multiple polyadenylation sites in the NAT1 gene. However, their role in NAT1 expression is poorly understood. Here, we have investigated the genetic sequence of the NAT1 gene in human cell lines, peripheral blood mononuclear cells and breast tumour tissue. We identified five potential polyadenylation signals, two of which carry known single nucleotide polymorphism that affect site usage. Cells that are homozygous for adenine at base 1642, the most distal polyadenylation site, use this site whereas those homozygous for cytosine at base 1642 could not. We also found that the presence of adenine at base 1642 is associated with the NAT1*10 haplotype. Because the putative binding site for miR-1290 is located between the last two polyadenylation sites, we hypothesised that cells that do not use the most distal site will be unaffected by miR-1290. However, this was not the case. NAT1 activity was positively correlated with miR-1290, and induction of miR-1290 in SH-SY5Y cells was associated with induction, not inhibition, of NAT1 activity. The use of PolyA1264 or PolyA1642 did not alter NAT1 activity following ectopic expression of a miR-1290 mimic. These results suggest that the role of miR-1290 in the regulation of NAT1 activity is more complex than previously reported.

Gene Annotation and Transcriptome Delineation on a De Novo Genome Assembly for the Reference Leishmania major Friedlin Strain.

Leishmania major is the main causative agent of cutaneous leishmaniasis in humans. The Friedlin strain of this species (LmjF) was chosen when a multi-laboratory consortium undertook the objective of deciphering the first genome sequence for a parasite of the genus Leishmania. The objective was successfully attained in 2005, and this represented a milestone for Leishmania molecular biology studies around the world. Although the LmjF genome sequence was done following a shotgun strategy and using classical Sanger sequencing, the results were excellent, and this genome assembly served as the reference for subsequent genome assemblies in other Leishmania species. Here, we present a new assembly for the genome of this strain (named LMJFC for clarity), generated by the combination of two high throughput sequencing platforms, Illumina short-read sequencing and PacBio Single Molecular Real-Time (SMRT) sequencing, which provides long-read sequences. Apart from resolving uncertain nucleotide positions, several genomic regions were reorganized and a more precise composition of tandemly repeated gene loci was attained. Additionally, the genome annotation was improved by adding 542 genes and more accurate coding-sequences defined for around two hundred genes, based on the transcriptome delimitation also carried out in this work. As a result, we are providing gene models (including untranslated regions and introns) for 11,238 genes. Genomic information ultimately determines the biology of every organism; therefore, our understanding of molecular mechanisms will depend on the availability of precise genome sequences and accurate gene annotations. In this regard, this work is providing an improved genome sequence and updated transcriptome annotations for the reference L. major Friedlin strain.

Identification of Alternative Polyadenylation in Cyanidioschyzon merolae Through Long-Read Sequencing of mRNA.

Alternative polyadenylation (APA) is widespread among metazoans and has been shown to have important impacts on mRNA stability and protein expression. Beyond a handful of well-studied organisms, however, its existence and consequences have not been well investigated. We therefore turned to the deep-branching red alga, Cyanidioschyzon merolae, to study the biology of polyadenylation in an organism highly diverged from humans and yeast. C. merolae is an acidothermophilic alga that lives in volcanic hot springs. It has a highly reduced genome (16.5 Mbp) and has lost all but 27 of its introns and much of its splicing machinery, suggesting that it has been under substantial pressure to simplify its RNA processing pathways. We used long-read sequencing to assess the key features of C. merolae mRNAs, including splicing status and polyadenylation cleavage site (PAS) usage. Splicing appears to be less efficient in C. merolae compared with yeast, flies, and mammalian cells. A high proportion of transcripts (63%) have at least two distinct PAS's, and 34% appear to utilize three or more sites. The apparent polyadenylation signal UAAA is used in more than 90% of cases, in cells grown in both rich media or limiting nitrogen. Our documentation of APA for the first time in this non-model organism highlights its conservation and likely biological importance of this regulatory step in gene expression.

A Genome-Wide Epstein-Barr Virus Polyadenylation Map and Its Antisense RNA to EBNA.

Epstein-Barr virus (EBV) is a ubiquitous human pathogen associated with Burkitt's lymphoma and nasopharyngeal carcinoma. Although the EBV genome harbors more than a hundred genes, a full transcription map with EBV polyadenylation profiles remains unknown. To elucidate the 3' ends of all EBV transcripts genome-wide, we performed the first comprehensive analysis of viral polyadenylation sites (pA sites) using our previously reported polyadenylation sequencing (PA-seq) technology. We identified that EBV utilizes a total of 62 pA sites in JSC-1, 60 in Raji, and 53 in Akata cells for the expression of EBV genes from both plus and minus DNA strands; 42 of these pA sites are commonly used in all three cell lines. The majority of identified pA sites were mapped to the intergenic regions downstream of previously annotated EBV open reading frames (ORFs) and viral promoters. pA sites lacking an association with any known EBV genes were also identified, mostly for the minus DNA strand within the EBNA locus, a major locus responsible for maintenance of viral latency and cell transformation. The expression of these novel antisense transcripts to EBNA were verified by 3' rapid amplification of cDNA ends (RACE) and Northern blot analyses in several EBV-positive (EBV+) cell lines. In contrast to EBNA RNA expressed during latency, expression of EBNA-antisense transcripts, which is restricted in latent cells, can be significantly induced by viral lytic infection, suggesting potential regulation of viral gene expression by EBNA-antisense transcription during lytic EBV infection. Our data provide the first evidence that EBV has an unrecognized mechanism that regulates EBV reactivation from latency.IMPORTANCE Epstein-Barr virus represents an important human pathogen with an etiological role in the development of several cancers. By elucidation of a genome-wide polyadenylation landscape of EBV in JSC-1, Raji, and Akata cells, we have redefined the EBV transcriptome and mapped individual polymerase II (Pol II) transcripts of viral genes to each one of the mapped pA sites at single-nucleotide resolution as well as the depth of expression. By unveiling a new class of viral lytic RNA transcripts antisense to latent EBNAs, we provide a novel mechanism of how EBV might control the expression of viral latent genes and lytic infection. Thus, this report takes another step closer to understanding EBV gene structure and expression and paves a new path for antiviral approaches.

Dysfunctional transcripts are formed by alternative polyadenylation in OPMD.

Post-transcription mRNA processing in the 3'-untranslated region (UTR) of transcripts alters mRNA landscape. Alternative polyadenylation (APA) utilization in the 3'-UTR often leads to shorter 3'-UTR affecting mRNA stability, a process that is regulated by PABPN1. In skeletal muscles PABPN1 levels reduce with age and a greater decrease in found in Oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant myopathy caused by expansion mutation in PABPN1. In OPMD models a shift from distal to proximal polyadenylation site utilization in the 3'-UTR, and PABPN1 was shown to play a prominent role in APA. Whether PABPN1-mediated APA transcripts are functional is not fully understood. We investigate nuclear export and translation efficiency of transcripts in OPMD models. We focused on autophagy-regulated genes (ATGs) with APA utilization in cell models with reduced functional PABPN1. We provide evidence that ATGs transcripts from distal PAS retain in the nucleus and thus have reduced translation efficiency in cells with reduced PABPN1. In contrast, transcripts from proximal PAS showed a higher cytoplasmic abundance but a reduced occupancy in the ribosome. We therefore suggest that in reduced PABPN1 levels ATG transcripts from APA may not effectively translate to proteins. In those conditions we found constitutive autophagosome fusion and reduced autophagy flux. Augmentation of PABPN1 restored autophagosome fusion, suggesting that PABPN1-mediated APA plays a role in autophagy in OPMD and in aging muscles.

Precursor microRNA-122 inhibits synthesis of Insig1 isoform mRNA by modulating polyadenylation site usage.

The insulin-induced gene 1 protein (Insig1) inhibits the cholesterol biosynthesis pathway by retaining transcription factor SREBP in the endoplasmic reticulum, and by causing the degradation of HMGCR, the rate-limiting enzyme in cholesterol biosynthesis. Liver-specific microRNA miR-122, on the other hand, enhances cholesterol biosynthesis by an unknown mechanism. We have found that Insig1 mRNAs are generated by alternative cleavage and polyadenylation, resulting in specific isoform mRNA species. During high cholesterol abundance, the short 1.4-kb Insig1 mRNA was found to be preferentially translated to yield Insig1 protein. Precursor molecules of miR-122 down-regulated the translation of the 1.4-kb Insig1 isoform mRNA by interfering with the usage of the promoter-proximal cleavage-polyadenylation site that gives rise to the 1.4-kb Insig1 mRNA. These findings argue that precursor miR-122 molecules modulate polyadenylation site usage in Insig1 mRNAs, resulting in down-regulation of Insig1 protein abundance. Thus, precursor microRNAs may have hitherto undetected novel functions in nuclear gene expression.

The epsilon motif of hepatitis B virus RNA exhibits a potassium-dependent ribonucleolytic activity.

Fourteen different classes of ribozymes are known that catalyse a range of diverse chemical reactions. We report here a novel potassium-dependent nucleolytic activity present in hepatitis B virus (HBV) RNA. A short RNA region (53 nt) with enzymatic properties released itself from the viral sequence by cis cleavages and could subsequently act in trans. The released region encompassed the epsilon motif present in the HBV RNA. The 3' end of the liberated fragment was within the noncanonical polyadenylation signal (UAUAAA) of the viral RNA while cleavages at about 53 nt upstream sites released the fragment. Mutations of the primary scissile sites or annealing these sites with the antisense oligodeoxyribonucleotides blocked the release of this short fragment and annulled subsequent trans cleavage activity. An exogenously synthesized short transcript of only this 53 nt was active as a sequence-independent trans-acting nuclease and cleaved after pyrimidines in viral or other substrate transcripts under physiological potassium ion concentrations. Formation of a G-quadruplex within this region was suggested by circular dichroism and nondenaturing polyacrylamide gel analyses. Our results reveal a unique natural example of a trans-acting ribonuclease that cleaves at multiple sites in a sequence-independent fashion. The presence of this novel activity implores a dynamic structural behaviour in the ε region and raises new questions about HBV gene regulation.

Overlooked FANCD2 variant encodes a promising, portent tumor suppressor, and alternative polyadenylation contributes to its expression.

Fanconi Anemia (FA) complementation group D2 protein (FANCD2) is the center of the FA tumor suppressor pathway, which has become an important field of investigation in human aging and cancer. Here we report an overlooked central player in the FA pathway, FANCD2 variant 2 (FANCD2-V2), which appears to perform more potent tumor suppressor-function compared to the known variant of FANCD2, namely, FANCD2-V1. Detailed analysis of the FANCD2 gene structure indicated a proximal and distal polyadenylation site (PAS), associated with V2 and V1 transcripts accordingly. RNA polymerase II Chromatin immunoprecipitation (ChIP) targeting the two PAS-regions determined lesser binding of RNA pol II to DNA fragments in the distal PAS region in non-malignant cells compared to malignant cells. Conversely, the opposite occurred in the proximal PAS region. Moreover, RNA immunoprecipitation (RIP) identified that U2 snRNP, a major component of RNA splicing complex that interacts with the 3'end of an intron, showed greater binding to the last intron of the FANCD2-V1 transcript in malignant cells compared to the non-malignant cells. Importantly, our data showed that in human tissue samples, the ratio of V2 /V1 expression in lung, bladder, or ovarian cancer correlates inversely with the tumor stages/grades. Therefore, these findings provide a previously unrecognized central player FANCD2-V2 and thus novel insights into human tumorigenesis, and indicate that V2/V1 can act as an effective biomarker in assisting the recognition of tumor malignance.

The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion.

Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells.

Global insights into alternative polyadenylation regulation.

Alternative pre-mRNA processing greatly increases the coding capacity of the human genome and regulatory factors involved in RNA processing play critical roles in tissue development and maintenance. Indeed, abnormal functions of RNA processing factors have been associated with a wide range of human diseases from cancer to neurodegenerative disorders. While many studies have emphasized the importance of alternative splicing (AS), recent high-throughput sequencing efforts have also allowed global surveys of alternative polyadenylation (APA). For the majority of pre-mRNAs, as well as some non-coding transcripts such as lncRNAs, APA selects different 3'-ends and thus modulates the availability of regulatory sites recognized by trans-acting regulatory effectors, including miRs and RNA binding proteins (RBPs). Here, we compare the available technologies for assessing global polyadenylation patterns, summarize the roles of auxiliary factors on APA, and discuss the impact of differential polyA site (pA) selection in the determination of cell fate, transformation and disease.

The polyadenylation code: a unified model for the regulation of mRNA alternative polyadenylation.

The majority of eukaryotic genes produce multiple mRNA isoforms with distinct 3' ends through a process called mRNA alternative polyadenylation (APA). Recent studies have demonstrated that APA is dynamically regulated during development and in response to environmental stimuli. A number of mechanisms have been described for APA regulation. In this review, we attempt to integrate all the known mechanisms into a unified model. This model not only explains most of previous results, but also provides testable predictions that will improve our understanding of the mechanistic details of APA regulation. Finally, we briefly discuss the known and putative functions of APA regulation.

TDP-43 regulates ß-adducin (Add2) transcript stability.

TDP-43 is an RNA-binding protein involved in several steps of mRNA metabolism including transcription, splicing and stability. It is also involved in ALS and FTD, neurodegenerative diseases characterized by TDP-43 nuclear depletion. We previously identified TDP-43 as a binder of the downstream element (DSE) of the ß-Adducin (Add2) brain-specific polyadenylation site (A4 PAS), suggesting its involvement in pre-mRNA 3' end processing. Here, by using chimeric minigenes, we showed that TDP-43 depletion in HeLa and HEK293 cells resulted in down-regulation of both the chimeric and endogenous Add2 transcripts. Despite having confirmed TDP-43-DSE in vitro interaction, we demonstrated that the in vivo effect was not mediated by the TDP-43-DSE interaction. In fact, substitution of the Add2 DSE with viral E-SV40 and L-SV40 DSEs, which are not TDP-43 targets, still resulted in decreased Add2 mRNA levels after TDP-43 downregulation. In addition, we failed to show interaction between TDP-43 and key polyadenylation factors, such as CstF-64 and CPSF160 and excluded TDP-43 involvement in pre-mRNA cleavage and regulation of polyA tail length. These evidences allowed us to exclude the pre-hypothesized role of TDP43 in modulating 3' end processing of Add2 pre-mRNA. Finally, we showed that TDP-43 regulates Add2 gene expression levels by increasing Add2 mRNA stability. Considering that Add2 in brain participates in synapse assembly, synaptic plasticity and their stability, and its genetic inactivation in mice leads to LTP, LTD, learning and motor-coordination deficits, we hypothesize that a possible loss of Add2 function by TDP-43 depletion may contribute to ALS and FTD disease states.

Dynamic expression of 3' UTRs revealed by Poisson hidden Markov modeling of RNA-Seq: implications in gene expression profiling.

RNA sequencing (RNA-Seq) allows for the identification of novel exon-exon junctions and quantification of gene expression levels. We show that from RNA-Seq data one may also detect utilization of alternative polyadenylation (APA) in 3' untranslated regions (3' UTRs) known to play a critical role in the regulation of mRNA stability, cellular localization and translation efficiency. Given the dynamic nature of APA, it is desirable to examine the APA on a sample by sample basis. We used a Poisson hidden Markov model (PHMM) of RNA-Seq data to identify potential APA in human liver and brain cortex tissues leading to shortened 3' UTRs. Over three hundred transcripts with shortened 3' UTRs were detected with sensitivity >75% and specificity >60%. Tissue-specific 3' UTR shortening was observed for 32 genes with a q-value ≤ 0.1. When compared to alternative isoforms detected by Cufflinks or MISO, our PHMM method agreed on over 100 transcripts with shortened 3' UTRs. Given the increasing usage of RNA-Seq for gene expression profiling, using PHMM to investigate sample-specific 3' UTR shortening could be an added benefit from this emerging technology.