Reciprocal intronic and exonic histone modification regions in humans.

While much attention has been focused on chromatin at promoters and exons, human genes are mostly composed of intronic sequences. Analyzing published surveys of nucleosomes and 41 chromatin marks in humans, we identified histone modifications specifically associated with 5' intronic sequences, distinguishable from promoter marks and bulk nucleosomes. These intronic marks were spatially reciprocal to trimethylated histone H3 Lys36 (H3K36me3), typically transitioning near internal exons. Several marks transitioned near bona fide exons, but not near nucleosomes at exon-like sequences. Therefore, we examined whether splicing affects histone marking. Even with considerable changes in regulated alternative splicing, histone marks were stable. Notably, these findings are consistent with exon definition influencing histone marks. In summary, we show that the location of many intragenic marks in humans can be distilled into a simple organizing principle: association with 5' intronic or 3' exonic regions

COMT genetic variation confers risk for psychotic and affective disorders: a case control study

BACKGROUND: Variation in the COMT gene has been implicated in a number of psychiatric disorders, including psychotic, affective and anxiety disorders. The majority of these studies have focused on the functional Val108/158Met polymorphism and yielded conflicting results, with limited studies examining the relationship between other polymorphisms, or haplotypes, and psychiatric illness. We hypothesized that COMT variation may confer a general risk for psychiatric disorders and have genotyped four COMT variants (Val158Met, rs737865, rs165599, and a SNP in the P2 promoter [-278A/G; rs2097603]) in 394 Caucasian cases and 467 controls. Cases included patients with schizophrenia (n = 196), schizoaffective disorder (n = 62), bipolar disorder (n = 82), major depression (n = 30), and patients diagnosed with either psychotic disorder NOS or depressive disorder NOS (n = 24). RESULTS: SNP rs2097603, the Val/Met variant and SNP rs165599 were significantly associated (p = 0.004; p = 0.05; p = 0.035) with a broad "all affected" diagnosis. Haplotype analysis revealed a potentially protective G-A-A-A haplotype haplotype (-278A/G; rs737865; Val108/158Met; rs165599), which was significantly underrepresented in this group (p = 0.0033) and contained the opposite alleles of the risk haplotype previously described by Shifman et al. Analysis of diagnostic subgroups within the "all affecteds group" showed an association of COMT in patients with psychotic disorders as well as in cases with affective illness although the associated variants differed. The protective haplotype remained significantly underrepresented in most of these subgroups. CONCLUSION: Our results support the view that COMT variation provides a weak general predisposition to neuropsychiatric disease including psychotic and affective disorders

Lineage Abundance Estimation for SARS-CoV-2 in Wastewater Using Transcriptome Quantification Techniques

Effectively monitoring the spread of SARS-CoV-2 mutants is essential to efforts to counter the ongoing pandemic. Predicting lineage abundance from wastewater, however, is technically challenging. We show that by sequencing SARS-CoV-2 RNA in wastewater and applying algorithms initially used for transcriptome quantification, we can estimate lineage abundance in wastewater samples. We find high variability in signal among individual samples, but the overall trends match those observed from sequencing clinical samples. Thus, while clinical sequencing remains a more sensitive technique for population surveillance, wastewater sequencing can be used to monitor trends in mutant prevalence in situations where clinical sequencing is unavailable

Diverse Forms of RPS9 Splicing Are Part of an Evolving Autoregulatory Circuit

Ribosomal proteins are essential to life. While the functions of ribosomal protein-encoding genes (RPGs) are highly conserved, the evolution of their regulatory mechanisms is remarkably dynamic. In Saccharomyces cerevisiae, RPGs are unusual in that they are commonly present as two highly similar gene copies and in that they are over-represented among intron-containing genes. To investigate the role of introns in the regulation of RPG expression, we constructed 16 S. cerevisiae strains with precise deletions of RPG introns. We found that several yeast introns function to repress rather than to increase steady-state mRNA levels. Among these, the RPS9A and RPS9B introns were required for cross-regulation of the two paralogous gene copies, which is consistent with the duplication of an autoregulatory circuit. To test for similar intron function in animals, we performed an experimental test and comparative analyses for autoregulation among distantly related animal RPS9 orthologs. Overexpression of an exogenous RpS9 copy in Drosophila melanogaster S2 cells induced alternative splicing and degradation of the endogenous copy by nonsense-mediated decay (NMD). Also, analysis of expressed sequence tag data from distantly related animals, including Homo sapiens and Ciona intestinalis, revealed diverse alternatively-spliced RPS9 isoforms predicted to elicit NMD. We propose that multiple forms of splicing regulation among RPS9 orthologs from various eukaryotes operate analogously to translational repression of the alpha operon by S4, the distant prokaryotic ortholog. Thus, RPS9 orthologs appear to have independently evolved variations on a fundamental autoregulatory circuit

Diverse forms of RPS9 splicing are part of an evolving autoregulatory circuit.

Ribosomal proteins are essential to life. While the functions of ribosomal protein-encoding genes (RPGs) are highly conserved, the evolution of their regulatory mechanisms is remarkably dynamic. In Saccharomyces cerevisiae, RPGs are unusual in that they are commonly present as two highly similar gene copies and in that they are over-represented among intron-containing genes. To investigate the role of introns in the regulation of RPG expression, we constructed 16 S. cerevisiae strains with precise deletions of RPG introns. We found that several yeast introns function to repress rather than to increase steady-state mRNA levels. Among these, the RPS9A and RPS9B introns were required for cross-regulation of the two paralogous gene copies, which is consistent with the duplication of an autoregulatory circuit. To test for similar intron function in animals, we performed an experimental test and comparative analyses for autoregulation among distantly related animal RPS9 orthologs. Overexpression of an exogenous RpS9 copy in Drosophila melanogaster S2 cells induced alternative splicing and degradation of the endogenous copy by nonsense-mediated decay (NMD). Also, analysis of expressed sequence tag data from distantly related animals, including Homo sapiens and Ciona intestinalis, revealed diverse alternatively-spliced RPS9 isoforms predicted to elicit NMD. We propose that multiple forms of splicing regulation among RPS9 orthologs from various eukaryotes operate analogously to translational repression of the alpha operon by S4, the distant prokaryotic ortholog. Thus, RPS9 orthologs appear to have independently evolved variations on a fundamental autoregulatory circuit

Hypothetical evolution of <i>RPS9</i> autoregulation.

<p>A) Hypothetical evolution of the <i>RPS9</i> autoregulatory circuit after duplication and divergence. Autoregulation of pre-WGD <i>RPS9</i> (top) is conserved between post-WGD gene copies despite divergence in expression levels to produce asymmetrical cross-regulation (middle). In <i>S. cerevisiae</i>, <i>RPS9A</i> and <i>RPS9B</i> intron deletions shift the burden of autoregulation onto the other intron-containing gene copy (bottom). B) A theoretical “biochemical toolkit,” which minimally requires an S4 RNA-binding domain and a suitable RNA binding site to perturb an essential step in gene expression (left), could potentially produce the many forms of splicing regulation observed in yeasts and animal <i>RPS9</i> orthologs (right).</p

Diverse alternatively spliced <i>RPS9</i> isoforms encode PTC+ exons associated with high nucleotide conservation and predicted RNA structures.

<p>Summaries of ESTs, predicted RNA structures, and sequence conservation from animal <i>RPS9</i> orthologs (<i>H. sapiens</i>, <i>X. tropicalis</i>, <i>O. latipes</i>, <i>D. melanogaster</i>, <i>C. intestinalis</i>, and <i>S. cerevisiae</i>) are presented along a dendogram illustrating their phylogenetic relationships (not to scale). For each species, histograms summarize EST coverage (gray bars) and inferred splice junctions with both 5′ GT (blue bars) and 3′ AG splice sites (red bars). Dashed lines separate the lower 5% and upper 95% histogram values; EST coverage is labeled on the y-axis. Two gene models (below each histogram) are plotted to scale (black line; 1 kb) representing either the major isoform (top gene model) or a spliced PTC+ EST (bottom gene model) for each species (an “unspliced” pre-mRNA is modeled for S. cerevisiae in lieu of an EST). The major isoform sequence is annotated as coding (thick black lines) or UTR (thin black lines) and interrupted by GT-AG introns (angled black lines). The first PTC (red line and octagon) in the representative PTC+ EST sequence (thin lines) is indicated. Below the two gene models, PhastCons scores (black bars), and RNAz predictions (green lines) indicate regions associated with high nucleotide conservation and statistically significant (P>0.9) RNA structure predictions, respectively (<i>X. tropicalis</i> not shown; <i>C. intestinalis</i> not applicable). PhastCons scores and RNAz predictions were based on MultiZ alignments obtained from the UCSC Genome Browser where available (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002620#s4" target="_blank">Methods</a>).</p

RPG intron deletions reveal gene-specific effects on steady-state mRNA levels.

<p>A–C) Microarray expression data for 16 RPG Δi mutants compared to a common wild-type strain. In each panel, the change in expression due to intron deletion is shown for either the intronless gene (red lines) or its paralogous gene copy (blue lines) compared to all other changes detected by microarray (boxplots). The effect of intron deletion is shown for each Δi mutant on A) the expression of the intronless gene copy, B) the expression of the paralogous gene copy, and C) the Intron Accumulation Index of the paralogous gene copy. Microarray data are expressed as the normalized log₂ transformed probe intensity for exon features averaged from at least two replicate microarrays. Whiskers represent 1.5 times the interquartile range. D) RT-qPCR quantification of <i>RPS9A</i> (red circles) and <i>RPS9B</i> (blue triangles) expression changes for each Δi mutant relative to wild-type (columns). <i>RPS9A</i> and <i>RPS9B</i> values were divided by SCR1 values to obtain ratios controlled for variations in cDNA quantity. Log₂ transformed ratios are plotted relative to wild-type (based on the mean of three biological replicates). Each of three biological replicates is shown as a point and the mean as a dash. E) The effect of intron deletion on the total number of transcripts encoding S9. Stacked barplots illustrate the percent of <i>RPS9A</i> (red bars) and <i>RPS9B</i> (blue bars) transcripts calculated for each Δi mutant. For a wild-type strain (first column), the percent of <i>RPS9A</i> and <i>RPS9B</i> transcripts encoding S9 were estimated from published RNA-seq data <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002620#pgen.1002620-Nagalakshmi1" target="_blank">[19]</a>. Changes in <i>RPS9A</i> and <i>RPS9B</i> transcript numbers for each Δi mutant (columns) were calculated by multiplying wild-type percentages by relative expression changes determined by qPCR.</p

<i>D. melanogaster RpS9</i> is autoregulated by alternative splicing coupled to NMD.

<p>A) Illustration of <i>RpS9</i> mRNA isoforms assessed by PCR (the PTC+ isoform is indicated by a red octagon). Primers sets (arrows) were designed to amplify multiple or specific <i>RpS9</i> mRNA isoforms (RT-PCR and qPCR primers, respectively). B) RT-PCR validation of the <i>RpS9</i> PTC+ mRNA isoform degraded by NMD. C) Experimental design used to assess the affect of <i>UPF1</i> knock-down on the abundance of <i>RpS9</i> mRNA isoforms. D) RT-qPCR determination of <i>RpS9</i> PTC+ mRNA isoform abundance (top panels) and total endogenous <i>RpS9</i> mRNA abundance (bottom panels) in S2 cells transfected with a plasmid constitutively expressing an <i>RpS9</i> cDNA (red circles) or an empty vector control (blue circles). The affect of <i>UPF1</i> knock-down (via incubation with dsRNA) on each <i>RpS9</i> mRNA isoform (right panels) is compared to a non-specific dsRNA control (left panels). <i>RpS9</i> mRNA isoform abundance values were divided by <i>GAPDH1</i> mRNA abundance values to obtain ratios internally controlled for variations in cDNA quantity. Log₂ transformed ratios for each of three biological replicates is shown as a point and the mean as a dash.</p