Gene expression, evolution, and the genetics of electrosensing in the smalltooth sawfish, Pristis pectinata.

Sawfishes (Pristidae) are large, highly threatened rays named for their tooth-studded rostrum, which is used for prey sensing and capture. Of all five species, the smalltooth sawfish, Pristis pectinata, has experienced the greatest decline in range, currently found in only ~20% of its historic range. To better understand the genetic underpinnings of these taxonomically and morphologically unique animals, we collected transcriptomic data from several tissue types, mapped them to the recently completed reference genome, and contrasted the patterns observed with comparable data from other elasmobranchs. Evidence of positive selection was detected in 79 genes in P. pectinata, several of which are involved in growth factor/receptor tyrosine kinase signaling and body symmetry and may be related to the unique morphology of sawfishes. Changes in these genes may impact cellular responses to environmental conditions such as temperature, dissolved oxygen, and salinity. Data acquired also allow for examination of the molecular components of P. pectinata electrosensory systems, which are highly developed in sawfishes and have likely been influential in their evolutionary success.

A genome-wide overexpression screen reveals Mycobacterium smegmatis growth inhibitors encoded by mycobacteriophage Hammy.

During infection, bacteriophages produce diverse gene products to overcome bacterial antiphage defenses, to outcompete other phages, and to take over cellular processes. Even in the best-studied model phages, the roles of most phage-encoded gene products are unknown, and the phage population represents a largely untapped reservoir of novel gene functions. Considering the sheer size of this population, experimental screening methods are needed to sort through the enormous collection of available sequences and identify gene products that can modulate bacterial behavior for downstream functional characterization. Here, we describe the construction of a plasmid-based overexpression library of 94 genes encoded by Hammy, a Cluster K mycobacteriophage closely related to those infecting clinically important mycobacteria. The arrayed library was systematically screened in a plate-based cytotoxicity assay, identifying a diverse set of 24 gene products (representing ∼25% of the Hammy genome) capable of inhibiting growth of the host bacterium Mycobacterium smegmatis. Half of these are related to growth inhibitors previously identified in related phage Waterfoul, supporting their functional conservation; the other genes represent novel additions to the list of known antimycobacterial growth inhibitors. This work, conducted as part of the HHMI-supported Science Education Alliance Gene-function Exploration by a Network of Emerging Scientists (SEA-GENES) project, highlights the value of parallel, comprehensive overexpression screens in exploring genome-wide patterns of phage gene function and novel interactions between phages and their hosts.

MiSiPi-Rna: an integrated tool for characterizing small regulatory RNA processing.

RNA interference (RNAi) is mediated by small (20-30 nucleotide) RNAs that are produced by complex processing pathways. In animals, three main classes are recognized: microRNAs (miRNAs), small-interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs). Understanding of small RNA pathways has benefited from genetic models where key enzymatic events were identified that lead to stereotypical positioning of small RNAs relative to precursor transcripts. Increasingly there is interest in using RNAi in non-model systems due to ease of generating synthetic small RNA precursors for research and biotechnology. Unfortunately, small RNAs are often rapidly evolving, requiring investigation of a species' endogenous small RNAs prior to deploying an RNAi approach. This can be accomplished through small non-coding RNA sequencing followed by applying various computational tools; however, the complexity and separately maintained packages lead to significant challenges for annotating global small RNA populations. To address this need, we developed a simple and efficient R package (MiSiPi-Rna) which can be used to characterize pre-selected loci with plots and statistics, aiding researchers understanding RNAi biology specific to their target species. Additionally, MiSiPi-Rna pioneers several computational approaches to identifying Dicer processing to assist annotation of miRNA and siRNA.