Local heat transfer on a finite width surface with laminar boundary layer flow [conference paper]

The effect of a lateral discontinuity in the thermal boundary conditions in two dimensional laminar flow on a flat plate, is investigated by numerical and analytical modeling. When the thermal and momentum boundary layers start at the same location, the resulting self-similar two dimensional boundary layer equations were solved numerically. For an unheated starting length, three dimensional numerical simulations were required. For both the three and two dimensional thermal simulations, a Blasius velocity field was assumed. It is found that all the Nusselt numbers collapse to a single curve when graphed as a function of a spanwise similarity variable. Simple correlations for the local Nusselt number on a rectangular flat plate are presented for a variety of boundary conditions

Local heat transfer on a finite width surface with laminar boundary layer flow [journal paper]

The effect of a lateral discontinuity in the thermal boundary conditions in two dimensional laminar flow on a flat plate is investigated with numerical and analytical modeling. When the thermal and momentum boundary layers start at the same location, the resulting self-similar two dimensional boundary layer equations were solved numerically. For flow with an unheated starting length, three dimensional numerical simulations were required. For both the two and three dimensional thermal simulations, the Blasius solution for a two dimensional momentum boundary layer was assumed. It is found that all the Nusselt numbers collapse to a single curve when graphed as a function of a spanwise similarity variable. Simple correlations for the local Nusselt number on a rectangular flat plate are presented for a variety of boundary conditions

Local heat transfer on a finite width surface with laminar boundary layer flow

The effect of a lateral discontinuity in the thermal boundary conditions in two dimensional laminar flow on a flat plate is investigated with numerical and analytical modeling. When the thermal and momentum boundary layers start at the same location, the resulting self-similar two dimensional boundary layer equations were solved numerically. For flow with an unheated starting length, three dimensional numerical simulations were required. For both the two and three dimensional thermal simulations, the Blasius solution for a two dimensional momentum boundary layer was assumed. It is found that all the Nusselt numbers collapse to a single curve when graphed as a function of a spanwise similarity variable. Simple correlations for the local Nusselt number on a rectangular flat plate are presented for a variety of boundary conditions

Evolution of a tissue-specific splicing network

Alternative splicing of precursor mRNA (pre-mRNA) is a strategy employed by most eukaryotes to increase transcript and proteomic diversity. Many metazoan splicing factors are members of multigene families, with each member having different functions. How these highly related proteins evolve unique properties has been unclear. Here we characterize the evolution and function of a new Drosophila splicing factor, termed LS2 (Large Subunit 2), that arose from a gene duplication event of dU2AF50, the large subunit of the highly conserved heterodimeric general splicing factor U2AF (U2-associated factor). The quickly evolving LS2 gene has diverged from the splicing-promoting, ubiquitously expressed dU2AF50 such that it binds a markedly different RNA sequence, acts as a splicing repressor, and is preferentially expressed in testes. Target transcripts of LS2 are also enriched for performing testes-related functions. We therefore propose a path for the evolution of a new splicing factor in Drosophila that regulates specific pre-mRNAs and contributes to transcript diversity in a tissue-specific manner

A Proteomics Approach to Profiling the Temporal Translational Response to Stress and Growth

Summary: To quantify dynamic protein synthesis rates, we developed MITNCAT, a method combining multiplexed isobaric mass tagging with pulsed SILAC (pSILAC) and bio-orthogonal non-canonical amino acid tagging (BONCAT) to label newly synthesized proteins with azidohomoalanine (Aha), thus enabling high temporal resolution across multiple conditions in a single analysis. MITNCAT quantification of protein synthesis rates following induction of the unfolded protein response revealed global down-regulation of protein synthesis, with stronger down-regulation of glycolytic and protein synthesis machinery proteins, but up-regulation of several key chaperones. Waves of temporally distinct protein synthesis were observed in response to epidermal growth factor, with altered synthesis detectable in the first 15 min. Comparison of protein synthesis with mRNA sequencing and ribosome footprinting distinguished protein synthesis driven by increased transcription versus increased translational efficiency. Temporal delays between ribosome occupancy and protein synthesis were observed and found to correlate with altered codon usage in significantly delayed proteins. : Functional Aspects of Cell Biology; Methodology in Biological Sciences; Proteomics Subject Areas: Functional Aspects of Cell Biology, Methodology in Biological Sciences, Proteomic

Halo‐seq: An RNA Proximity Labeling Method for the Isolation and Analysis of Subcellular RNA Populations

The subcellular localization of specific RNA molecules promotes localized cellular activity across a variety of species and cell types. The misregulation of this RNA targeting can result in developmental defects, and mutations in proteins that regulate this process are associated with multiple diseases. For the vast majority of localized RNAs, however, the mechanisms that underlie their subcellular targeting are unknown, partly due to the difficulty associated with profiling and quantifying subcellular RNA populations. To address this challenge, we developed Halo-seq, a proximity labeling technique that can label and profile local RNA content at virtually any subcellular location. Halo-seq relies on a HaloTag fusion protein localized to a subcellular space of interest. Through the use of a radical-producing Halo ligand, RNAs that are near the HaloTag fusion are specifically labeled with spatial and temporal control. Labeled RNA is then specifically biotinylated in vitro via a click reaction, facilitating its purification from a bulk RNA sample using streptavidin beads. The content of the biotinylated RNA is then profiled using high-throughput sequencing. In this article, we describe the experimental and computational procedures for Halo-seq, including important benchmark and quality control steps. By allowing the flexible profiling of a variety of subcellular RNA populations, we envision Halo-seq facilitating the discovery and further study of RNA localization regulatory mechanisms. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Visualization of HaloTag fusion protein localization Basic Protocol 2: In situ copper-catalyzed cycloaddition of fluorophore via click reaction Basic Protocol 3: In vivo RNA alkynylation and extraction of total RNA Basic Protocol 4: In vitro copper-catalyzed cycloaddition of biotin via click reaction Basic Protocol 5: Assessment of RNA biotinylation by RNA dot blot Basic Protocol 6: Enrichment of biotinylated RNA using streptavidin beads and preparation of RNA-seq library Basic Protocol 7: Computational analysis of Halo-seq data

ELAV/Hu RNA binding proteins determine multiple programs of neural alternative splicing.

ELAV/Hu factors are conserved RNA binding proteins (RBPs) that play diverse roles in mRNA processing and regulation. The founding member, Drosophila Elav, was recognized as a vital neural factor 35 years ago. Nevertheless, little was known about its impacts on the transcriptome, and potential functional overlap with its paralogs. Building on our recent findings that neural-specific lengthened 3' UTR isoforms are co-determined by ELAV/Hu factors, we address their impacts on splicing. While only a few splicing targets of Drosophila are known, ectopic expression of each of the three family members (Elav, Fne and Rbp9) alters hundreds of cassette exon and alternative last exon (ALE) splicing choices. Reciprocally, double mutants of elav/fne, but not elav alone, exhibit opposite effects on both classes of regulated mRNA processing events in larval CNS. While manipulation of Drosophila ELAV/Hu RBPs induces both exon skipping and inclusion, characteristic ELAV/Hu motifs are enriched only within introns flanking exons that are suppressed by ELAV/Hu factors. Moreover, the roles of ELAV/Hu factors in global promotion of distal ALE splicing are mechanistically linked to terminal 3' UTR extensions in neurons, since both processes involve bypass of proximal polyadenylation signals linked to ELAV/Hu motifs downstream of cleavage sites. We corroborate the direct action of Elav in diverse modes of mRNA processing using RRM-dependent Elav-CLIP data from S2 cells. Finally, we provide evidence for conservation in mammalian neurons, which undergo broad programs of distal ALE and APA lengthening, linked to ELAV/Hu motifs downstream of regulated polyadenylation sites. Overall, ELAV/Hu RBPs orchestrate multiple broad programs of neuronal mRNA processing and isoform diversification in Drosophila and mammalian neurons