Regulatory Expansion in Mammals of Multivalent hnRNP Assemblies that Globally Control Alternative Splicing.

Alternative splicing (AS) patterns have diverged rapidly during vertebrate evolution, yet the functions of most species- and lineage-specific splicing events are not known. We observe that mammalian-specific AS events are enriched in transcript sequences encoding intrinsically disordered regions (IDRs) of proteins, in particular those containing glycine/tyrosine repeats that mediate formation of higher-order protein assemblies implicated in gene regulation and human disease. These evolutionary changes impact nearly all members of the hnRNP A and D families of RNA binding proteins. Regulation of these events requires formation of unusual, long-range mammalian-specific RNA duplexes. Differential inclusion of the alternative exons controls the formation of tyrosine-dependent multivalent hnRNP assemblies that, in turn, function to globally regulate splicing. Together, our results demonstrate that AS control of IDR-mediated interactions between hnRNPs represents an important and recurring mechanism underlying splicing regulation. Furthermore, this mechanism has expanded the regulatory capacity of mammalian cells.

Rad5 Recruits Error-Prone DNA Polymerases for Mutagenic Repair of ssDNA Gaps on Undamaged Templates.

Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA base lesions in both an error-free and an error-prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. We find that PRR responds to DNA replication stress that does not cause base lesions. Rad5 forms nuclear foci during normal S phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the sites of stressed DNA replication forks, where it recruits TLS polymerases to repair single-stranded DNA (ssDNA) gaps, preventing mitotic defects and chromosome breaks. In contrast to the prevailing view of PRR, our data indicate that Rad5 promotes both mutagenic and error-free repair of undamaged ssDNA that arises during physiological and exogenous replication stress.

Coding regions affect mRNA stability in human cells.

A new paradigm has emerged that coding regions can regulate mRNA stability in model organisms. Here, due to differences in cognate tRNA abundance, synonymous codons are translated at different speeds, and slow codons then stimulate mRNA decay. To ask if this phenomenon also occurs in humans, we isolated RNA stability effects due to coding regions using the human ORFeome collection. We find that many open reading frame (ORF) characteristics, such as length and secondary structure, fail to provide explanations for how coding regions alter mRNA stability, and, instead, that the ORF relies on translation to impact mRNA stability. Consistent with what has been seen in other organisms, codon use is related to the effects of ORFs on transcript stability. Importantly, we found instability-associated codons have longer A-site dwell times, suggesting for the first time in humans a connection between elongation speed and mRNA decay. Thus, we propose that codon usage alters decoding speeds and so affects human mRNA stability.

Structural basis for the regulatory role of the PPxY motifs in the thioredoxin-interacting protein TXNIP.

TXNIP (thioredoxin-interacting protein) negatively regulates the antioxidative activity of thioredoxin and participates in pleiotropic cellular processes. Its deregulation is linked to various human diseases, including diabetes, acute myeloid leukaemia and cardiovascular diseases. The E3 ubiquitin ligase Itch (Itchy homologue) polyubiquitinates TXNIP to promote its degradation via the ubiquitin-proteasome pathway, and this Itch-mediated polyubiquitination of TXNIP is dependent on the interaction of the four WW domains of Itch with the two PPxY motifs of TXNIP. However, the molecular mechanism of this interaction of TXNIP with Itch remains elusive. In the present study, we found that each of the four WW domains of Itch exhibited different binding affinities for TXNIP, whereas multivalent engagement between the four WW domains of Itch and the two PPxY motifs of TXNIP resulted in their strong binding avidity. Our structural analyses demonstrated that the third and fourth WW domains of Itch were able to recognize both PPxY motifs of TXNIP simultaneously, supporting a multivalent binding mode between Itch and TXNIP. Interestingly, the phosphorylation status on the tyrosine residue of the PPxY motifs of TXNIP serves as a molecular switch in its choice of binding partners and thereby downstream biological signalling outcomes. Phosphorylation of this tyrosine residue of TXNIP diminished the binding capability of PPxY motifs of TXNIP to Itch, whereas this phosphorylation is a prerequisite to the binding activity of TXNIP to SHP2 [SH2 (Src homology 2) domain-containing protein tyrosine phosphatase 2] and their roles in stabilizing the phosphorylation and activation of CSK (c-Src tyrosine kinase).

Variations and inequities in access to cardiac diagnostic services in Ontario Canada.

OBJECTIVES: Echocardiography is an essential diagnostic modality known to have wide regional utilization variations. This study's objectives were to quantify regional variations and to examine the extent to which they are explained by differences in population age, sex, cardiac disease prevalence (CDP), and social determinants of health (SDH) risk. METHODS: This is an observational study of all echocardiography exams performed in Ontario in 2019/20 (n = 695,622). We measured regional variations in echocardiography crude rates and progressively standardized rates for population age, sex, CDP, and SDH risk. RESULTS: After controlling for differences in population age, sex, and CDP, Ontario's highest rate regions had echocardiography rates 57% higher than its lowest rate regions. Forty eight percent of total variation was not explained by differences in age, sex, and CDP. CDP increased with SDH risk. Access to most cardiac diagnostics was negatively correlated with SDH risk, while cardiac catheterization rates were positively correlated with SDH risk. CONCLUSION: Variations analysis that adjusts for age and sex only without including clinical measures of need are likely to overestimate the unwarranted portion of total variation. Substantial variations persisted despite a mandatory provider accreditation policy aimed at curtailing them. The associations between variations and SDH risks imply a need to redress access and outcome inequities.

Transcriptional buffering and 3'UTR lengthening are shaped during human neurodevelopment by shifts in mRNA stability and microRNA load.

The contribution of mRNA half-life is commonly overlooked when examining changes in mRNA abundance during development. mRNA levels of some genes are regulated by transcription rate only, but others may be regulated by mRNA half-life only shifts. Furthermore, transcriptional buffering is predicted when changes in transcription rates have compensating shifts in mRNA half-life resulting in no change to steady-state levels. Likewise, transcriptional boosting should result when changes in transcription rate are accompanied by amplifying half-life shifts. During neurodevelopment there is widespread 3'UTR lengthening that could be shaped by differential shifts in the stability of existing short or long 3'UTR transcript isoforms. We measured transcription rate and mRNA half-life changes during induced human Pluripotent Stem Cell (iPSC)-derived neuronal development using RATE-seq. During transitions to progenitor and neuron stages, transcriptional buffering occurred in up to 50%, and transcriptional boosting in up to 15%, of genes with changed transcription rates. The remaining changes occurred by transcription rate only or mRNA half-life only shifts. Average mRNA half-life decreased two-fold in neurons relative to iPSCs. Short gene isoforms were more destabilized in neurons and thereby increased the average 3'UTR length. Small RNA sequencing captured an increase in microRNA copy number per cell during neurodevelopment. We propose that mRNA destabilization and 3'UTR lengthening are driven in part by an increase in microRNA load in neurons. Our findings identify mRNA stability mechanisms in human neurodevelopment that regulate gene and isoform level abundance and provide a precedent for similar post-transcriptional regulatory events as other tissues develop.

Buffering of transcription rate by mRNA half-life is a conserved feature of Rett syndrome models.

Transcriptional changes in Rett syndrome (RTT) are assumed to directly correlate with steady-state mRNA levels, but limited evidence in mice suggests that changes in transcription can be compensated by post-transcriptional regulation. We measure transcription rate and mRNA half-life changes in RTT patient neurons using RATEseq, and re-interpret nuclear and whole-cell RNAseq from Mecp2 mice. Genes are dysregulated by changing transcription rate or half-life and are buffered when both change. We utilized classifier models to predict the direction of transcription rate changes and find that combined frequencies of three dinucleotides are better predictors than CA and CG. MicroRNA and RNA-binding Protein (RBP) motifs are enriched in 3'UTRs of genes with half-life changes. Nuclear RBP motifs are enriched on buffered genes with increased transcription rate. We identify post-transcriptional mechanisms in humans and mice that alter half-life or buffer transcription rate changes when a transcriptional modulator gene is mutated in a neurodevelopmental disorder.

Mitochondrial control through nutritionally regulated global histone H3 lysine-4 demethylation.

Histone demethylation by Jumonji-family proteins is coupled with the decarboxylation of α-ketoglutarate (αKG) to yield succinate, prompting hypotheses that their activities are responsive to levels of these metabolites in the cell. Consistent with this paradigm we show here that the Saccharomyces cerevisiae Jumonji demethylase Jhd2 opposes the accumulation of H3K4me3 in fermenting cells only when they are nutritionally manipulated to contain an elevated αKG/succinate ratio. We also find that Jhd2 opposes H3K4me3 in respiratory cells that do not exhibit such an elevated αKG/succinate ratio. While jhd2∆ caused only limited gene expression defects in fermenting cells, transcript profiling and physiological measurements show that JHD2 restricts mitochondrial respiratory capacity in cells grown in non-fermentable carbon in an H3K4me-dependent manner. In association with these phenotypes, we find that JHD2 limits yeast proliferative capacity under physiologically challenging conditions as measured by both replicative lifespan and colony growth on non-fermentable carbon. JHD2's impact on nutrient response may reflect an ancestral role of its gene family in mediating mitochondrial regulation.