Finding differentially expressed sRNA-Seq regions with srnadiff

Small RNAs (sRNAs) encompass a great variety of molecules of different kinds, such as microRNAs, small interfering RNAs, Piwi-associated RNA, among others. These sRNAs have a wide range of activities, which include gene regulation, protection against virus, transposable element silencing, and have been identified as a key actor in determining the development of the cell. Small RNA sequencing is thus routinely used to assess the expression of the diversity of sRNAs, usually in the context of differentially expression, where two conditions are compared. Tools that detect differentially expressed microRNAs are numerous, because microRNAs are well documented, and the associated genes are well defined. However, tools are lacking to detect other types of sRNAs, which are less studied, and whose precursor RNA is not well characterized. We present here a new method, called srnadiff, which finds all kinds of differentially expressed sRNAs. To the extent of our knowledge, srnadiff is the first tool that detects differentially expressed sRNAs without the use of external information, such as genomic annotation or additional sequences of sRNAs.

Splicing factor mutations in hematologic malignancies

Mutations in genes encoding RNA splicing factors were discovered nearly ten years ago and are now understood to be amongst the most recurrent genetic abnormalities in patients with all forms of myeloid neoplasms and several types of lymphoproliferative disorders as well as subjects with clonal hematopoiesis. These discoveries implicate aberrant RNA splicing, the process by which precursor RNA is converted into mature messenger RNA, in the development of clonal hematopoietic conditions. Both the protein as well as the RNA components of the splicing machinery are affected by mutations at highly specific residues and a number of these mutations alter splicing in a manner distinct from loss of function. Importantly, cells bearing these mutations have now been shown to generate mRNA species with novel aberrant sequences, some of which may be critical to disease pathogenesis and/or novel targets for therapy. These findings have opened new avenues of research to understand biological pathways disrupted by altered splicing. In parallel, multiple studies have revealed that cells bearing change-of-function mutation in splicing factors are preferentially sensitized to any further genetic or chemical perturbations of the splicing machinery. These discoveries are now being pursued in several early phase clinical trials using molecules with diverse mechanisms of action. Here we review the molecular effects of splicing factor mutations on splicing, mechanisms by which these mutations drive clonal transformation of hematopoietic cells, and the development of new therapeutics targeting these genetic subsets of hematopoietic malignancies.

The RNA-binding protein RBP45D of Arabidopis plays a role in epigenetic control of flowering time and DCL3-independent RNA-directed DNA methylation

RNA-directed DNA methylation (RdDM) helps to defend plants against invasive nucleic acids. In the canonical form of RdDM, 24-nt small interfering RNAs (siRNAs) are produced by DICER-LIKE 3 (DCL3). Here, we describe the Arabidopsis thaliana prors1 (LUC) transgenic system, in which transcriptional gene silencing (TGS) is independent of DLC3. A forward genetics screen performed with this system identified both known components of RdDM, and the RNA-binding protein RBP45D. RBP45D promotes DNA methylation, and its loss delays flowering, especially at high temperature, presumably mediated by elevated FLC levels. RBP45D is localized to the nucleus, where it is associated with snRNAs and snoRNAs. RBP45D maintains siRNA production originating from the LUC transgene, but does not alter mRNA levels or affect processing of transcripts of known RdDM genes. We suggest that RBPD45 facilitates DCL3-independent siRNA production by stabilising either the precursor RNA or the as yet unidentified slicer protein.

RNAi-Mediated Control of Lepidopteran Pests of Important Crop Plants

Insects as pests destroy annually an estimated 18–20% of the crop production worldwide. Caterpillars, the larval stage of moths, are the major pests of agricultural products owing to their voracious feeding habits. In the past few decades, the potent methods of insect control, such as insecticides and Bt toxins, have been constrained as a result of health hazards, environmental issues, and development of resistance, after their prolonged application. Thus, there is need to find alternative options to improve plant protection strategies. Recently, RNA interference (RNAi), the post-transcriptional gene-silencing mechanism, has emerged as one of such a novel, sustainable, and environment friendly approaches for insect management and crop protection. RNAi technology relies on selection of a vital insect pest target gene and its expression as a double stranded RNA or stem-loop RNA molecule, which is recognized by the host RNAi machinery and processed into small interfering RNAs (siRNAs) or microRNAs (miRNAs). The siRNA/miRNA along with the RNA-induced silencing complex (RISC) binds to the complimentary mRNA and induce gene silencing at post-transcriptional level. With effective target-gene selection and transgenic plants expressing these precursor RNA molecules, insect pests of various crops have been efficiently managed. In this chapter, we discuss the basic mechanism of RNAi and its application in controlling lepidopteran pests of important crop plants.

Cofactors are Remnants of Life’s Origin and Early Evolution

The RNA World is one of the most widely accepted hypotheses explaining the origin of the genetic system used by all organisms today. It proposes that the tripartite system of DNA, RNA, and proteins was preceded by one consisting solely of RNA, which both stored genetic information and performed the molecular functions encoded by that genetic information. Current research into a potential RNA World revolves around the catalytic properties of RNA-based enzymes, or ribozymes. Well before the discovery of ribozymes, Harold White proposed that evidence for a precursor RNA world could be found within modern proteins in the form of coenzymes, the majority of which contain nucleobases or nucleoside moieties, such as Coenzyme A and S-adenosyl methionine, or are themselves nucleotides, such as ATP and NADH (a dinucleotide). These coenzymes, White suggested, had been the catalytic active sites of ancient ribozymes, which transitioned to their current forms after the surrounding ribozyme scaffolds had been replaced by protein apoenzymes during the evolution of translation. Since its proposal four decades ago, this groundbreaking hypothesis has garnered support from several different research disciplines and motivated similar hypotheses about other classes of cofactors, most notably iron-sulfur cluster cofactors as remnants of the geochemical setting of the origin of life. Evidence from prebiotic geochemistry, ribozyme biochemistry, and evolutionary biology, increasingly supports these hypotheses. Certain coenzymes and cofactors may bridge modern biology with the past and can thus provide insights into the elusive and poorly-recorded period of the origin and early evolution of life.

RJunBase: a database of RNA splice junctions in human normal and cancerous tissues

Splicing is an essential step of RNA processing for multi-exon genes, in which introns are removed from a precursor RNA, thereby producing mature RNAs containing splice junctions. Here, we develope the RJunBase (www.RJunBase.org), a web-accessible database of three types of RNA splice junctions (linear, back-splice, and fusion junctions) that are derived from RNA-seq data of non-cancerous and cancerous tissues. The RJunBase aims to integrate and characterize all RNA splice junctions of both healthy or pathological human cells and tissues. This new database facilitates the visualization of the gene-level splicing pattern and the junction-level expression profile, as well as the demonstration of unannotated and tumor-specific junctions. The first release of RJunBase contains 682 017 linear junctions, 225 949 back-splice junctions and 34 733 fusion junctions across 18 084 non-cancerous and 11 540 cancerous samples. RJunBase can aid researchers in discovering new splicing-associated targets and provide insights into the identification and assessment of potential neoepitopes for cancer treatment.

Preferential production of RNA rings by T4 RNA ligase 2 without any splint through rational design of precursor strand

Rings of single-stranded RNA are promising for many practical applications, but the methods to prepare them in preparative scale have never been established. Previously, RNA circularization was achieved by T4 RNA ligase 2 (Rnl2, a dsRNA ligase) using splints, but the yield was low due to concurrent intermolecular polymerization. Here, various functional RNAs (siRNA, miRNA, ribozyme, etc.) are dominantly converted by Rnl2 to the rings without significant limitations in sizes and sequences. The key is to design a precursor RNA, which is highly activated for the efficient circularization without any splint. First, secondary structure of target RNA ring is simulated by Mfold, and then hypothetically cut at one site so that a few intramolecular base pairs are formed at the terminal. Simply by treating this RNA with Rnl2, the target ring was selectively and efficiently produced. Unexpectedly, circular RNA can be obtained in high yield (>90%), even when only 2 bp form in the 3′-OH side and no full match base pair forms in the 5′-phosphate side. Formation of polymeric by-products was further suppressed by diluting conventional Rnl2 buffer to abnormally low concentrations. Even at high-RNA concentrations (e.g. 50 μM), enormously high selectivity (>95%) was accomplished.

An Orphan CpG Island Drives Expression of a let-7 miRNA Precursor with an Important Role in Mouse Development

Most human genes are associated with promoters embedded in non-methylated, G + C-rich CpG islands (CGIs). Not all CGIs are found at annotated promoters, however, raising the possibility that many serve as promoters for transcripts that do not code for proteins. To test this hypothesis, we searched for novel transcripts in embryonic stem cells (ESCs) that originate within orphan CGIs. Among several candidates, we detected a transcript that included three members of the let-7 micro-RNA family: Let-7a-1, let-7f-1, and let-7d. Deletion of the CGI prevented expression of the precursor RNA and depleted the included miRNAs. Mice homozygous for this mutation were sub-viable and showed growth and other defects. The results suggest that despite the identity of their seed sequences, members of the let-7 miRNA family exert distinct functions that cannot be complemented by other members.

Mining of miRNAs using Next Generation Sequencing (NGS) data generated for Okra (Abelmoschus esculentus)

MicroRNAs (miRNAs) are small, highly conserved non-coding RNA molecules involved in theregulation of gene expression in eukaryotes. Gene expression involves post-transcriptionalgene regulation by miRNAs. miRNAs are formed from precursor RNA molecules that fold intoa stem loop secondary structure. The mature miRNA is one end of the precursor miRNA,defined by the cut from ‘Drosha’ on either the 5’ or 3’ arm. In this study, we have used abioinformatics approach to identify miRNAs in 3,361 contigs obtained from partial genomesequence data of Abelmoschus esculentus (okra) sequenced by NGS technology. Using C-miiand psRNA Target tools, we identified two miRNAs and their target RNAs for which a regulatorymiRNA binding has been verified. Their targets consisted of transcription factors involved ingrowth and development, gene regulation and metabolism. Phylogenetic analysis of the newlyidentified miRNA family has been done to compare their level of conservation with respect tothe other members of the plant kingdom.