Polyadenylation Site Usage Research Articles

RAP: RNA-Seq Analysis Pipeline, a new cloud-based NGS web application.

BackgroundThe study of RNA has been dramatically improved by the introduction of Next Generation Sequencing platforms allowing massive and cheap sequencing of selected RNA fractions, also providing information on strand orientation (RNA-Seq). The complexity of transcriptomes and of their regulative pathways make RNA-Seq one of most complex field of NGS applications, addressing several aspects of the expression process (e.g. identification and quantification of expressed genes and transcripts, alternative splicing and polyadenylation, fusion genes and trans-splicing, post-transcriptional events, etc.).Moreover, the huge volume of data generated by NGS platforms introduces unprecedented computational and technological challenges to efficiently analyze and store sequence data and results.MethodsIn order to provide researchers with an effective and friendly resource for analyzing RNA-Seq data, we present here RAP (RNA-Seq Analysis Pipeline), a cloud computing web application implementing a complete but modular analysis workflow. This pipeline integrates both state-of-the-art bioinformatics tools for RNA-Seq analysis and in-house developed scripts to offer to the user a comprehensive strategy for data analysis. RAP is able to perform quality checks (adopting FastQC and NGS QC Toolkit), identify and quantify expressed genes and transcripts (with Tophat, Cufflinks and HTSeq), detect alternative splicing events (using SpliceTrap) and chimeric transcripts (with ChimeraScan). This pipeline is also able to identify splicing junctions and constitutive or alternative polyadenylation sites (implementing custom analysis modules) and call for statistically significant differences in genes and transcripts expression, splicing pattern and polyadenylation site usage (using Cuffdiff2 and DESeq).ResultsThrough a user friendly web interface, the RAP workflow can be suitably customized by the user and it is automatically executed on our cloud computing environment. This strategy allows to access to bioinformatics tools and computational resources without specific bioinformatics and IT skills. RAP provides a set of tabular and graphical results that can be helpful to browse, filter and export analyzed data, according to the user needs.

Single-cell polyadenylation site mapping reveals 3' isoform choice variability.

Cell-to-cell variability in gene expression is important for many processes in biology, including embryonic development and stem cell homeostasis. While heterogeneity of gene expression levels has been extensively studied, less attention has been paid to mRNA polyadenylation isoform choice. 3′ untranslated regions regulate mRNA fate, and their choice is tightly controlled during development, but how 3′ isoform usage varies within genetically and developmentally homogeneous cell populations has not been explored. Here, we perform genome-wide quantification of polyadenylation site usage in single mouse embryonic and neural stem cells using a novel single-cell transcriptomic method, BATSeq. By applying BATBayes, a statistical framework for analyzing single-cell isoform data, we find that while the developmental state of the cell globally determines isoform usage, single cells from the same state differ in the choice of isoforms. Notably this variation exceeds random selection with equal preference in all cells, a finding that was confirmed by RNA FISH data. Variability in 3′ isoform choice has potential implications on functional cell-to-cell heterogeneity as well as utility in resolving cell populations.

Global 3' UTR shortening has a limited effect on protein abundance in proliferating T cells.

Alternative polyadenylation is a cellular mechanism that generates mRNA isoforms differing in their 3' untranslated regions (3' UTRs). Changes in polyadenylation site usage have been described upon induction of proliferation in resting cells, but the underlying mechanism and functional significance of this phenomenon remain largely unknown. To understand the functional consequences of shortened 3' UTR isoforms in a physiological setting, we used 3' end sequencing and quantitative mass spectrometry to determine polyadenylation site usage, mRNA and protein levels in murine and human naive and activated T cells. Although 3' UTR shortening in proliferating cells is conserved between human and mouse, orthologous genes do not exhibit similar expression of alternative 3' UTR isoforms. We generally find that 3' UTR shortening is not accompanied by a corresponding change in mRNA and protein levels. This suggests that although 3' UTR shortening may lead to changes in the RNA-binding protein interactome, it has limited effects on protein output.

Alternative polyadenylation regulates CELF1/CUGBP1 target transcripts following T cell activation

Alternative polyadenylation (APA) is an evolutionarily conserved mechanism for regulating gene expression. Transcript 3′ end shortening through changes in polyadenylation site usage occurs following T cell activation, but the consequences of APA on gene expression are poorly understood. We previously showed that GU-rich elements (GREs) found in the 3′ untranslated regions of select transcripts mediate rapid mRNA decay by recruiting the protein CELF1/CUGBP1. Using a global RNA sequencing approach, we found that a network of CELF1 target transcripts involved in cell division underwent preferential 3′ end shortening via APA following T cell activation, resulting in decreased inclusion of CELF1 binding sites and increased transcript expression. We present a model whereby CELF1 regulates APA site selection following T cell activation through reversible binding to nearby GRE sequences. These findings provide insight into the role of APA in controlling cellular proliferation during biological processes such as development, oncogenesis and T cell activation.

SLaP mapper: A webserver for identifying and quantifying spliced-leader addition and polyadenylation site usage in kinetoplastid genomes

The Kinetoplastida are a diverse and globally distributed class of free-living and parasitic single-celled eukaryotes that collectively cause a significant burden on human health and welfare. In kinetoplastids individual genes do not have promoters, but rather all genes are arranged downstream of a small number of RNA polymerase II transcription initiation sites and are thus transcribed in polycistronic gene clusters. Production of individual mRNAs from this continuous transcript occurs co-transcriptionally by trans-splicing of a ∼39 nucleotide capped RNA and subsequent polyadenylation of the upstream mRNA. SLaP mapper (Spliced-Leader and Polyadenylation mapper) is a fully automated web-service for identification, quantitation and gene-assignment of both spliced-leader and polyadenylation addition sites in Kinetoplastid genomes. SLaP mapper only requires raw read data from paired-end Illumina RNAseq and performs all read processing, mapping, quality control, quantification, and analysis in a fully automated pipeline. To provide usage examples and estimates of the quantity of sequence data required we use RNAseq obtained from two different library preparations from both Trypanosoma brucei and Leishmania mexicana to show the number of expected reads that are obtained from each preparation type. SLaP mapper is an easy to use, platform independent webserver that is freely available for use at http://www.stevekellylab.com/software/slap. Example files are provided on the website.

ZNF143 is regulated through alternative 3'UTR isoforms.

ZNF143 is a ubiquitously expressed transcription factor conserved in all vertebrates, regulating genes involved in primary metabolism and cell growth. It is therefore crucial to tightly maintain the adequate level of this factor in the cell. Although ZNF143 expression is auto-regulated at the transcriptional level, nothing is known about the post-transcriptional events influencing its expression. In this work, performed in mammalian cells, we show that ZNF143 expresses different 3'-untranslated regions (3'-UTR) asa result of alternative polyadenylation. These 3'UTR isoforms have a diverse impact on the ZNF143 transcript fate. Indeed, we show that the longest isoform, unlike the short one, contains a destabilizingAU-Rich element and is targeted by the miRNA 590-3p. Additionally we observed a correlation between ZNF143 downregulation and miR-590-3p up-regulation in retinoic acid treated teratocarcinoma cells. This strongly suggests that ZNF143 post-transcriptional regulation depends on the long 3'UTR isoform during teratocarcinoma cells differentiation. Finally we evidenced that the alternative polyadenylation site usage is independent of the previously identified ZNF143 transcriptional auto-regulation.

Diversity and dynamics of the Drosophila transcriptome.

Animal transcriptomes are dynamic, each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. We identified new genes, transcripts, and proteins using poly(A)+ RNA sequence from Drosophila melanogaster cultured cell lines, dissected organ systems, and environmental perturbations. We found a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long noncoding RNAs (lncRNAs) some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized arising from combinatorial usage of promoters, splice sites, and polyadenylation sites.

A Novel Feed-Forward Loop between ARIH2 E3-Ligase and PABPN1 Regulates Aging-Associated Muscle Degeneration

Alanine expansion mutations in poly(A)-binding protein nuclear 1 (PABPN1) cause muscle weakness in the late-onset disorder oculopharyngeal muscular dystrophy. In affected muscles, expanded PABPN1 forms nuclear aggregates, depleting levels of soluble PABPN1 and inducing a genome-wide shift from distal to proximal polyadenylation site usage. PABPN1 protein accumulation is regulated by the ubiquitin proteasome system, which is highly dysregulated in oculopharyngeal muscular dystrophy. We show that ARIH2 E3-ligase regulates PABPN1 protein accumulation and aggregation. Levels of ARIH2 mRNA are regulated by PABPN1 via proximal polyadenylation site usage. We demonstrate that masking the proximal polyadenylation site in ARIH2 3' untranslated region by antisense oligonucleotides elevates the expression of ARIH2 and PABPN1 and restores myogenic defects that are induced by ARIH2 or PABPN1 down-regulation in cell culture. Invivo ARIH2 mRNA levels significantly decrease from midlife in vastus lateralis muscles and highly correlate with muscle degeneration. We suggest that the expression of both genes is maintained by a feed-forward loop between mRNA stability regulated by PABPN1 and protein turnover regulated by ARIH2.

The nucleosome regulates the usage of polyadenylation sites in the human genome

BackgroundIt has been reported that 3' end processing is coupled to transcription and nucleosome depletion near the polyadenylation sites in many species. However, the association between nucleosome occupancy and polyadenylation site usage is still unclear.ResultsBy systematic analysis of high-throughput sequencing datasets from the human genome, we found that nucleosome occupancy patterns are different around the polyadenylation sites, and that the patterns associate with both transcription termination and recognition of polyadenylation sites. Upstream of proximal polyadenylation sites, RNA polymerase II accumulated and nucleosomes were better positioned compared with downstream of the sites. Highly used proximal polyadenylation sites had higher upstream nucleosome levels and RNA polymerase II accumulation than lowly used sites. This suggests that nucleosomes positioned upstream of proximal sites function in the recognition of proximal polyadenylation sites and in the preparation for 3' end processing by slowing down transcription speed. Both conserved distal polyadenylation sites and constitutive sites showed stronger nucleosome depletion near polyadenylation sites and had intrinsically better positioned downstream nucleosomes. Finally, there was a higher accumulation of RNA polymerase II downstream of the polyadenylation sites, to guarantee gene transcription termination and recognition of the last polyadenylation sites, if previous sites were missed.ConclusionsOur study indicates that nucleosome arrays play different roles in the regulation of the usage of polyadenylation sites and transcription termination of protein-coding genes, and form a dual pausing model of RNA polymerase II in the alternative polyadenylation sites’ region, to ensure effective 3' end processing.

DeepSAGE Reveals Genetic Variants Associated with Alternative Polyadenylation and Expression of Coding and Non-coding Transcripts

Many disease-associated variants affect gene expression levels (expression quantitative trait loci, eQTLs) and expression profiling using next generation sequencing (NGS) technology is a powerful way to detect these eQTLs. We analyzed 94 total blood samples from healthy volunteers with DeepSAGE to gain specific insight into how genetic variants affect the expression of genes and lengths of 3′-untranslated regions (3′-UTRs). We detected previously unknown cis-eQTL effects for GWAS hits in disease- and physiology-associated traits. Apart from cis-eQTLs that are typically easily identifiable using microarrays or RNA-sequencing, DeepSAGE also revealed many cis-eQTLs for antisense and other non-coding transcripts, often in genomic regions containing retrotransposon-derived elements. We also identified and confirmed SNPs that affect the usage of alternative polyadenylation sites, thereby potentially influencing the stability of messenger RNAs (mRNA). We then combined the power of RNA-sequencing with DeepSAGE by performing a meta-analysis of three datasets, leading to the identification of many more cis-eQTLs. Our results indicate that DeepSAGE data is useful for eQTL mapping of known and unknown transcripts, and for identifying SNPs that affect alternative polyadenylation. Because of the inherent differences between DeepSAGE and RNA-sequencing, our complementary, integrative approach leads to greater insight into the molecular consequences of many disease-associated variants.

U1snRNP-mediated suppression of polyadenylation in conjunction with the RNA structure controls poly (A) site selection in foamy viruses

BackgroundDuring reverse transcription, retroviruses duplicate the long terminal repeats (LTRs). These identical LTRs carry both promoter regions and functional polyadenylation sites. To express full-length transcripts, retroviruses have to suppress polyadenylation in the 5′LTR and activate polyadenylation in the 3′LTR. Foamy viruses have a unique LTR structure with respect to the location of the major splice donor (MSD), which is located upstream of the polyadenylation signal.ResultsHere, we describe the mechanisms of foamy viruses regulating polyadenylation. We show that binding of the U1 small nuclear ribonucleoprotein (U1snRNP) to the MSD suppresses polyadenylation at the 5′LTR. In contrast, polyadenylation at the 3′LTR is achieved by adoption of a different RNA structure at the MSD region, which blocks U1snRNP binding and furthers RNA cleavage and subsequent polyadenylation.ConclusionRecently, it was shown that U1snRNP is able to suppress the usage of intronic cryptic polyadenylation sites in the cellular genome. Foamy viruses take advantage of this surveillance mechanism to suppress premature polyadenylation at the 5’end of their RNA. At the 3’end, Foamy viruses use a secondary structure to presumably block access of U1snRNP and thereby activate polyadenylation at the end of the genome. Our data reveal a contribution of U1snRNP to cellular polyadenylation site selection and to the regulation of gene expression.

Quantification of stochastic noise of splicing and polyadenylation in Entamoeba histolytica

Alternative splicing and polyadenylation were observed pervasively in eukaryotic messenger RNAs. These alternative isoforms could either be consequences of physiological regulation or stochastic noise of RNA processing. To quantify the extent of stochastic noise in splicing and polyadenylation, we analyzed the alternative usage of splicing and polyadenylation sites in Entamoeba histolytica using RNA-Seq. First, we identified a large number of rarely spliced alternative junctions and then showed that the occurrence of these alternative splicing events is correlated with splicing site sequence, occurrence of constitutive splicing events and messenger RNA abundance. Our results implied the majority of these alternative splicing events are likely to be stochastic error of splicing machineries, and we estimated the corresponding error rates. Second, we observed extensive microheterogeneity of polyadenylation cleavage sites, and the extent of such microheterogeneity is correlated with the occurrence of constitutive cleavage events, suggesting most of such microheterogeneity is likely to be stochastic. Overall, we only observed a small fraction of alternative splicing and polyadenylation isoforms that are unlikely to be solely stochastic, implying the functional relevance of alternative splicing and polyadenylation in E. histolytica is limited. Lastly, we revised the gene models and annotated their 3′UTR in AmoebaDB, providing valuable resources to the community.

IGF-I 3' untranslated region: strain-specific polymorphisms and motifs regulating IGF-I in osteoblasts.

Reduced IGF-I is associated with low bone mass in humans and mice. C3H/He/J (C3H) mice have higher skeletal IGF-I and greater bone mass than C57BL/6J (B6). We hypothesized that strain-related genotypic differences in Igf1 affected skeletal function. The Igf1 coding region is nonpolymorphic, but its 3' untranslated region (UTR) is polymorphic between C3H and B6. Luciferase-Igf1 3' UTR reporter constructs showed that these polymorphic regions did not affect UTR function. IGF-I splice variants give rise to a common mature IGF-I peptide, but different E peptides. We identified two splice products, exon 4+6 (Ea) and exon 4+5+6 (Eb, mechano-growth factor) and found that their abundance was unchanged during osteoblastic differentiation. The Igf1 3' UTR encoded by exon 6 contains alternative polyadenylation sites. Proximal site use produces a short 3' UTR of approximately 195 bases, whereas distal site usage results in an approximately 6300-base UTR. Although Igf1 mRNA levels did not change during osteoblastic differentiation, distal polyadenylation site usage was increased in B6 cells but not in C3H. The resulting long Igf1 RNA isoform is less stable and has decreased translation efficiency, which may be one mechanism contributing to decreased IGF-I in B6 vs. C3H mice. Although the long UTR contains a conserved [GU](18) repeat, which is a positive regulator of UTR activity, it is also targeted by negative regulators, miR-29 and miR-365. These microRNAs are increased in B6 and C3H cells during osteoblastic differentiation. Differential expression of the long Igf1 3' UTR isoform may be a possible mechanism for enhanced IGF-I regulation in B6 vs. C3H mice.

Poly(A) binding protein nuclear 1 levels affect alternative polyadenylation

The choice for a polyadenylation site determines the length of the 3′-untranslated region (3′-UTRs) of an mRNA. Inclusion or exclusion of regulatory sequences in the 3′-UTR may ultimately affect gene expression levels. Poly(A) binding protein nuclear 1 (PABPN1) is involved in polyadenylation of pre-mRNAs. An alanine repeat expansion in PABPN1 (exp-PABPN1) causes oculopharyngeal muscular dystrophy (OPMD). We hypothesized that previously observed disturbed gene expression patterns in OPMD muscles may have been the result of an effect of PABPN1 on alternative polyadenylation, influencing mRNA stability, localization and translation. A single molecule polyadenylation site sequencing method was developed to explore polyadenylation site usage on a genome-wide level in mice overexpressing exp-PABPN1. We identified 2012 transcripts with altered polyadenylation site usage. In the far majority, more proximal alternative polyadenylation sites were used, resulting in shorter 3′-UTRs. 3′-UTR shortening was generally associated with increased expression. Similar changes in polyadenylation site usage were observed after knockdown or overexpression of expanded but not wild-type PABPN1 in cultured myogenic cells. Our data indicate that PABPN1 is important for polyadenylation site selection and that reduced availability of functional PABPN1 in OPMD muscles results in use of alternative polyadenylation sites, leading to large-scale deregulation of gene expression.

Genome-wide Analysis of Pre-mRNA 3′ End Processing Reveals a Decisive Role of Human Cleavage Factor I in the Regulation of 3′ UTR Length

Through alternative polyadenylation, human mRNAs acquire longer or shorter 3' untranslated regions, the latter typically associated with higher transcript stability and increased protein production. To understand the dynamics of polyadenylation site usage, we performed transcriptome-wide mapping of both binding sites of 3' end processing factors CPSF-160, CPSF-100, CPSF-73, CPSF-30, Fip1, CstF-64, CstF-64τ, CF I(m)25, CF I(m)59, and CF I(m)68 and 3' end processing sites in HEK293 cells. We found that although binding sites of these factors generally cluster around the poly(A) sites most frequently used in cleavage, CstF-64/CstF-64τ and CFI(m) proteins have much higher positional specificity compared to CPSF components. Knockdown of CF I(m)68 induced a systematic use of proximal polyadenylation sites, indicating that changes in relative abundance of a single 3' end processing factor can modulate the length of 3' untranslated regions across the transcriptome and suggesting a mechanism behind the previously observed increase in tumor cell invasiveness upon CF I(m)68 knockdown.

Human N-acetyltransferase 1 *10 and *11 alleles increase protein expression through distinct mechanisms and associate with sulfamethoxazole-induced hypersensitivity

N-acetyltransferase 1 (NAT1) metabolizes drugs and environmental carcinogens. NAT1 alleles *10 and *11 have been proposed to alter protein level or enzyme activity compared with wild-type NAT1 *4 and to confer cancer risk, through uncertain pathways. This study characterizes regulatory polymorphisms and underlying mechanisms of NAT1 expression. We measured allelic NAT1 mRNA expression and translation, as a function of multiple transcription start sites, alternative splicing, and three 3'-polyadenylation sites in human livers (one of which was discovered in this study), B lymphocytes, and transfected cells. In a clinical study of 469 patients with HIV/AIDS treated with the NAT1/NAT2 substrate sulfamethoxazole (SMX), associations were tested between SMX-induced hypersensitivity and NAT1 *10 and *11 genotypes, together with known NAT2 polymorphisms. NAT1 *10 and *11 were determined to act as common regulatory alleles accounting for most NAT1 expression variability, both leading to increased translation into active protein. NAT1 *11 (2.4% minor allele frequency) affected 3'-polyadenylation site usage, thereby increasing formation of NAT1 mRNA with intermediate length 3'-untranslated region (major isoform) at the expense of the short isoform, resulting in more efficient protein translation. NAT1 *10 (19% minor allele frequency) increased translation efficiency without affecting 3'-untranslated region polyadenylation site usage. Livers and B-lymphocytes with *11/*4 and *10/*10 genotypes displayed higher NAT1 immunoreactivity and NAT1 enzyme activity than the reference genotype *4/*4. Patients who carry *10/*10 and *11/*4 (fast NAT1 acetylators) were less likely to develop hypersensitivity to SMX, but this was observed only in individuals who are also carrying a slow NAT2 acetylator genotype. NAT1 *10 and *11 significantly increase NAT1 protein level/enzyme activity, enabling the classification of carriers into reference and rapid acetylators. Rapid NAT1 acetylator status seems to protect against SMX toxicity by compensating for slow NAT2 acetylator status.

RNA-binding protein HuR autoregulates its expression by promoting alternative polyadenylation site usage

RNA-binding protein HuR modulates the stability and translational efficiency of messenger RNAs (mRNAs) encoding essential components of the cellular proliferation, growth and survival pathways. Consistent with these functions, HuR levels are often elevated in cancer cells and reduced in senescent and quiescent cells. However, the molecular mechanisms that control HuR expression are poorly understood. Here we show that HuR protein autoregulates its abundance through a negative feedback loop that involves interaction of the nuclear HuR protein with a GU-rich element (GRE) overlapping with the HuR major polyadenylation signal (PAS2). An increase in the cellular HuR protein levels stimulates the expression of long HuR mRNA species containing an AU-rich element (ARE) that destabilizes the mRNAs and thus reduces the protein production output. The PAS2 read-through occurs due to a reduced recruitment of the CstF-64 subunit of the pre-mRNA cleavage stimulation factor in the presence of the GRE-bound HuR. We propose that this mechanism maintains HuR homeostasis in proliferating cells. Since only the nuclear HuR is expected to contribute to the auto-regulation, our model may explain the longstanding observation that the increase in the total HuR expression in cancer cells often correlates with the accumulation of its substantial fraction in the cytoplasm.

Differential usage of transcriptional start sites and polyadenylation sites in FMR1 premutation alleles

5′- and 3′-untranslated regions (UTRs) are important regulators of gene expression and play key roles in disease progression and susceptibility. The 5′-UTR of the fragile X mental retardation 1 (FMR1) gene contains a CGG repeat element that is expanded (>200 CGG repeats; full mutation) and methylated in fragile X syndrome (FXS), the most common form of inherited intellectual disability (ID) and known cause of autism. Significant phenotypic involvement has also emerged in some individuals with the premutation (55–200 CGG repeats), including fragile X-associated premature ovarian insufficiency (FXPOI) in females, and the neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS), in older adult carriers. Here, we show that FMR1 mRNA in human and mouse brain is expressed as a combination of multiple isoforms that use alternative transcriptional start sites and different polyadenylation sites. Furthermore, we have identified a novel human transcription start site used in brain but not in lymphoblastoid cells, and have detected FMR1 isoforms generated through the use of both canonical and non-canonical polyadenylation signals. Importantly, in both human and mouse, a specific regulation of the UTRs is observed in brain of FMR1 premutation alleles, suggesting that the transcript variants may play a role in premutation-related pathologies.

Common alleles in human N‐acetyltransferase 1 (NAT1 *10 and *11) increase enzyme activity via distinct mechanisms and associate with sulfamethoxazole induced hypersensitivity

N‐acetyltransferase 1 (NAT1) metabolizes drugs and environmental carcinogens, affecting drug response and cancer susceptibility. Two common NAT1 alleles, *10 and *11, have been reported to alter enzyme activity compared to wild‐type NAT1 (*4), but their effects and underlying mechanisms remain uncertain. We measured allelic NAT1 mRNA expression and translation, considering multiple transcription start sites, alternative splicing, and three 3′‐polyadenylation sites in human livers and B lymphocytes. *11 was found to favor usage of the second 3'polyadenylation site, increasing formation of NAT1 mRNA with the 3'UTR of intermediate length (major isoform). This major 3'UTR isoform supported more efficient protein translation than the short 3'UTR isoform, resulting in increased *11 NAT1 enzyme activity. Similarly, NAT1 *10 also increased protein translation efficiency, but without affecting NAT1 mRNA levels or 3′‐UTR polyadenylation site usage. B‐lymphocytes with *11/*4 and *10/*10 genotypes displayed higher NAT1 enzyme activity than the reference genotype *4/*4. Moreover, carriers of *10/*10 and *11/*4 (‘fast NAT1 acetylators') were less likely to develop hypersensitivity to NAT1/NAT2 substrate drug sulfamethoxazole, but this was observed only in subjects with slow NAT2 acetylator genotype. In conclusion, NAT1 *10 and *11 significantly increase NAT1 enzyme activity, enabling the classification of carriers into normal and rapid acetylators. Supported by NIH grant R21 AI074399 and U01 GM092655.

Most “Dark Matter” Transcripts Are Associated With Known Genes

Author SummaryThe human genome was sequenced a decade ago, but its exact gene composition remains a subject of debate. The number of protein-coding genes is much lower than initially expected, and the number of distinct transcripts is much larger than the number of protein-coding genes. Moreover, the proportion of the genome that is transcribed in any given cell type remains an open question: results from “tiling” microarray analyses suggest that transcription is pervasive and that most of the genome is transcribed, whereas new deep sequencing-based methods suggest that most transcripts originate from known genes. We have addressed this discrepancy by comparing samples from the same tissues using both technologies. Our analyses indicate that RNA sequencing appears more reliable for transcripts with low expression levels, that most transcripts correspond to known genes or are near known genes, and that many transcripts may represent new exons or aberrant products of the transcription process. We also identify several thousand small transcripts that map outside known genes; their sequences are often conserved and are often encoded in regions of open chromatin. We propose that most of these transcripts may be by-products of the activity of enhancers, which associate with promoters as part of their role as long-range gene regulatory sites. Overall, however, we find that most of the genome is not appreciably transcribed.