Computational workflow for investigating highly variable genes in single-cell RNA-seq across multiple time points and cell types.

Here, we present a computational approach for investigating highly variable genes (HVGs) associated with biological pathways of interest, across multiple time points and cell types in single-cell RNA-sequencing (scRNA-seq) data. Using public dengue virus and COVID-19 datasets, we describe steps for using the framework to characterize the dynamic expression levels of HVGs related to common and cell-type-specific biological pathways over multiple immune cell types. For complete details on the use and execution of this protocol, please refer to Arora et al.1

Antisense oligonucleotide induction of the hnRNPA1b isoform affects pre-mRNA splicing of SMN2 in SMA type I fibroblasts

Spinal muscular atrophy (SMA) is a severe, debilitating neuromuscular condition characterised by loss of motor neurons and progressive muscle wasting. SMA is caused by a loss of expression of SMN1 that encodes the survival motor neuron (SMN) protein necessary for the survival of motor neurons. Restoration of SMN expression through increased inclusion of SMN2 exon 7 is known to ameliorate symptoms in SMA patients. As a consequence, regulation of pre-mRNA splicing of SMN2 could provide a potential molecular therapy for SMA. In this study, we explored if splice switching antisense oligonucleotides could redirect the splicing repressor hnRNPA1 to the hnRNPA1b isoform and restore SMN expression in fibroblasts from a type I SMA patient. Antisense oligonucleotides (AOs) were designed to promote exon 7b retention in the mature mRNA and induce the hnRNPA1b isoform. RT-PCR and western blot analysis were used to assess and monitor the efficiency of different AO combinations. A combination of AOs targeting multiple silencing motifs in hnRNPA1 pre-mRNA led to robust hnRNPA1b induction, which, in turn, significantly increased expression of full-length SMN (FL-SMN) protein. A combination of PMOs targeting the same motifs also strongly induced hnRNPA1b isoform, but surprisingly SMN2 exon 5 skipping was detected, and the PMO cocktail did not lead to a significant increase in expression of FL-SMN protein. We further performed RNA sequencing to assess the genome-wide effects of hnRNPA1b induction. Some 3244 genes were differentially expressed between the hnRNPA1b-induced and untreated SMA fibroblasts, which are functionally enriched in cell cycle and chromosome segregation processes. RT-PCR analysis demonstrated that expression of the master regulator of these enrichment pathways, MYBL2 and FOXM1B, were reduced in response to PMO treatment. These findings suggested that induction of hnRNPA1b can promote SMN protein expression, but not at sufficient levels to be clinically relevant

Single-cell temporal analysis of natural dengue infection reveals skin-homing lymphocyte expansion one day before defervescence.

Effective clinical management of acute dengue virus (DENV) infection relies on the timing of suitable treatments during the disease progression. We analyzed single-cell transcriptomic profiles of the peripheral blood mononuclear cell samples from two DENV patients, collected daily during acute phase and also at convalescence. Key immune cell types demonstrated different dynamic responses over the course of the infection. On the day before defervescence (Day -1), we observed the peak expression of several prominent genes in the adaptive immunological pathways. We also characterized unique effector T cell clusters that expressed skin-homing signature genes at Day -1, whereas upregulation of skin and gut homing genes was also observed in plasma cells and plasmablasts during the febrile period. This work provides an overview of unique molecular dynamics that signify the entry of the critical phase, and the findings could improve the patient management of DENV infection

Transcriptional regulation of the ambient temperature response by h2a.z nucleosomes and hsf1 transcription factors in arabidopsis

Temperature influences the distribution, range, and phenology of plants. The key transcriptional activators of heat shock response in eukaryotes, the heat shock factors (HSFs), have undergone large-scale gene amplification in plants. While HSFs are central in heat stress responses, their role in the response to ambient temperature changes is less well understood. We show here that the warm ambient temperature transcriptome is dependent upon the HSFA1 clade of Arabidopsis HSFs, which cause a rapid and dynamic eviction of H2A.Z nucleosomes at target genes. A transcriptional cascade results in the activation of multiple downstream stress-responsive transcription factors, triggering large-scale changes to the transcriptome in response to elevated temperature. H2A.Z nucleosomes are enriched at temperature-responsive genes at non-inducible temperature, and thus likely confer inducibility of gene expression and higher responsive dynamics. We propose that the antagonistic effects of H2A.Z and HSF1 provide a mechanism to activate gene expression rapidly and precisely in response to temperature, while preventing leaky transcription in the absence of an activation signal.Biological and Soft Matter Physic