Single-cell RNA-sequencing of PBMCs from SAVI patients reveals disease-associated monocytes with elevated integrated stress response.

Gain-of-function mutations in stimulator of interferon gene 1 (STING1) result in STING-associated vasculopathy with onset in infancy (SAVI), a severe autoinflammatory disease. Although elevated type I interferon (IFN) production is thought to be the leading cause of the symptoms observed in patients, STING can induce a set of pathways, which have roles in the onset and severity of SAVI and remain to be elucidated. To this end, we performed a multi-omics comparative analysis of peripheral blood mononuclear cells (PBMCs) and plasma from SAVI patients and healthy controls, combined with a dataset of healthy PBMCs treated with IFN-ß. Our data reveal a subset of disease-associated monocyte, expressing elevated CCL3, CCL4, and IL-6, as well as a strong integrated stress response, which we suggest is the result of direct PERK activation by STING. Cell-to-cell communication inference indicates that these monocytes lead to T cell early activation, resulting in their senescence and apoptosis. Last, we propose a transcriptomic signature of STING activation, independent of type I IFN response.

Post-transcriptional polyadenylation site cleavage maintains 3'-end processing upon DNA damage.

The recognition of polyadenylation signals (PAS) in eukaryotic pre-mRNAs is usually coupled to transcription termination, occurring while pre-mRNA is chromatin-bound. However, for some pre-mRNAs, this 3'-end processing occurs post-transcriptionally, i.e., through a co-transcriptional cleavage (CoTC) event downstream of the PAS, leading to chromatin release and subsequent PAS cleavage in the nucleoplasm. While DNA-damaging agents trigger the shutdown of co-transcriptional chromatin-associated 3'-end processing, specific compensatory mechanisms exist to ensure efficient 3'-end processing for certain pre-mRNAs, including those that encode proteins involved in the DNA damage response, such as the tumor suppressor p53. We show that cleavage at the p53 polyadenylation site occurs in part post-transcriptionally following a co-transcriptional cleavage event. Cells with an engineered deletion of the p53 CoTC site exhibit impaired p53 3'-end processing, decreased mRNA and protein levels of p53 and its transcriptional target p21, and altered cell cycle progression upon UV-induced DNA damage. Using a transcriptome-wide analysis of PAS cleavage, we identify additional pre-mRNAs whose PAS cleavage is maintained in response to UV irradiation and occurring post-transcriptionally. These findings indicate that CoTC-type cleavage of pre-mRNAs, followed by PAS cleavage in the nucleoplasm, allows certain pre-mRNAs to escape 3'-end processing inhibition in response to UV-induced DNA damage.

A2Sign: Agnostic Algorithms for Signatures-a universal method for identifying molecular signatures from transcriptomic datasets prior to cell-type deconvolution.

MOTIVATION: Molecular signatures are critical for inferring the proportions of cell types from bulk transcriptomics data. However, the identification of these signatures is based on a methodology that relies on prior biological knowledge of the cell types being studied. When working with less known biological material, a data-driven approach is required to uncover the underlying classes and generate ad hoc signatures from healthy or pathogenic tissue. RESULTS: We present a new approach, A2Sign: Agnostic Algorithms for Signatures, based on a non-negative tensor factorization (NTF) strategy that allows us to identify cell-type-specific molecular signatures, greatly reduce collinearities and also account for inter-individual variability. We propose a global framework that can be applied to uncover molecular signatures for cell-type deconvolution in arbitrary tissues using bulk transcriptome data. We also present two new molecular signatures for deconvolution of up to 16 immune cell types using microarray or RNA-seq data. AVAILABILITY AND IMPLEMENTATION: All steps of our analysis were implemented in annotated Python notebooks (https://github.com/paulfogel/A2SIGN). To perform NTF, we used the NMTF package, which can be downloaded using Python pip install. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The Novel Phosphate and Bile Acid Sequestrant Polymer SAR442357 Delays Disease Progression in a Rat Model of Diabetic Nephropathy.

As a gut-restricted, nonabsorbed therapy, polymeric bile acid sequestrants (BAS) play an important role in managing hyperlipidemia and hyperglycemia. Similarly, nonabsorbable sequestrants of dietary phosphate have been used for the management of hyperphosphatemia in end-stage renal disease. To evaluate the potential utility of such polymer sequestrants to treat type 2 diabetes (T2D) and its associated renal and cardiovascular complications, we synthesized a novel polymeric sequestrant, SAR442357, possessing optimized bile acid (BA) and phosphate sequestration characteristics. Long-term treatment of T2D obese cZucker fatty/Spontaneously hypertensive heart failure F1 hybrid (ZSF1) with SAR442357 resulted in enhanced sequestration of BAs and phosphate in the gut, improved glycemic control, lowering of serum cholesterol, and attenuation of diabetic kidney disease (DKD) progression. In comparison, colesevelam, a BAS with poor phosphate binding properties, did not prevent DKD progression, whereas losartan, an angiotensin II receptor blocker that is widely used to treat DKD, showed no effect on hyperglycemia. Analysis of hepatic gene expression levels of the animals treated with SAR442357 revealed upregulation of genes responsible for the biosynthesis of cholesterol and BAs, providing clear evidence of target engagement and mode of action of the new sequestrant. Additional hepatic gene expression pathway changes were indicative of an interruption of the enterohepatic BA cycle. Histopathological analysis of ZSF1 rat kidneys treated with SAR442357 further supported its nephroprotective properties. Collectively, these findings reveal the pharmacological benefit of simultaneous sequestration of BAs and phosphate in treating T2D and its associated comorbidities and cardiovascular complications. SIGNIFICANCE STATEMENT: A new nonabsorbed polymeric sequestrant with optimum phosphate and bile salt sequestration properties was developed as a treatment option for DKD. The new polymeric sequestrant offered combined pharmacological benefits including glucose regulation, lipid lowering, and attenuation of DKD progression in a single therapeutic agent.

Regulation of RNA polymerase III transcription during transformation of human IMR90 fibroblasts with defined genetic elements.

RNA polymerase (Pol) III transcribes small untranslated RNAs that are essential for cellular homeostasis and growth. Its activity is regulated by inactivation of tumor suppressor proteins and overexpression of the oncogene c-MYC, but the concerted action of these tumor-promoting factors on Pol III transcription has not yet been assessed. In order to comprehensively analyse the regulation of Pol III transcription during tumorigenesis we employ a model system that relies on the expression of five genetic elements to achieve cellular transformation. Expression of these elements in six distinct transformation intermediate cell lines leads to the inactivation of TP53, RB1, and protein phosphatase 2A, as well as the activation of RAS and the protection of telomeres by TERT, thereby conducting to full tumoral transformation of IMR90 fibroblasts. Transformation is accompanied by moderately enhanced levels of a subset of Pol III-transcribed RNAs (7SK; MRP; H1). In addition, mRNA and/or protein levels of several Pol III subunits and transcription factors are upregulated, including increased protein levels of TFIIIB and TFIIIC subunits, of SNAPC1 and of Pol III subunits. Strikingly, the expression of POLR3G and of SNAPC1 is strongly enhanced during transformation in this cellular transformation model. Collectively, our data indicate that increased expression of several components of the Pol III transcription system accompanied by a 2-fold increase in steady state levels of a subset of Pol III RNAs is sufficient for sustaining tumor formation.

General transcription factors and subunits of RNA polymerase III: Paralogs for promoter- and cell type-specific transcription in multicellular eukaryotes.

In the course of evolution of multi-cellular eukaryotes, paralogs of general transcription factors and RNA polymerase subunits emerged. Paralogs of transcription factors and of the RPC32 subunit of RNA polymerase III play important roles in cell type- and promoter-specific transcription. Here we discuss their respective functions.

Using profiles based on nucleotide hydrophobicity to define essential regions for splicing.

The splice-site sequences of U2-type introns are highly degenerate, so many different sequences can function as U2-type splice sites. Using our new profiles based on hydrophobicity properties we pointed out specific properties for regions surrounding splice sites. We built a set T of flanking regions of genes with 1-3 introns from 21st and 22nd chromosomes extracted from GenBank to define positions having conserved properties, namely hydrophobicity, that are potentially essential for recognition by spliceosome. GT-AG introns exist in U2 and U12-types. Therefore, intron type cannot be simply determined by the dinucleotide termini. We attempted to distinguish U2 and U12-types introns with help of hydrophobicity profiles on sets of spice sites for U2 or U12-type introns extracted from SpliceRack database. The positions given by our method, which may be important for recognition by spliceosome, were compared to the nucleotide consensus provided by a classical method, Pictogram. We showed that there is a similarity of hydrophobicity profiles inside intron types. On one hand, GT-AG and GC-AG introns belonging to U2-type have resembling hydrophobicity profiles as well as AT-AC and GT-AG introns belonging to U12-type. On the other hand, hydrophobicity profiles of U2 and U12-types GT-AG introns are completely different. We suggest that hydrophobicity profiles facilitate definition of intron type, distinguishing U2 and U12 intron types and can be used to build programs to search splice site and to evaluate their strength. Therefore, our study proves that hydrophobicity profiles are informative method providing insights into mechanisms of splice sites recognition.