ABSTRACT
The vertebrate-conserved RNA-binding protein DND1 is required for the survival of primordial germ cells (PGCs), as well as the suppression of germ cell tumours in mice. Here we show that in mice DND1 binds a UU(A/U) trinucleotide motif predominantly in the 3' untranslated regions of mRNA, and destabilizes target mRNAs through direct recruitment of the CCR4-NOT deadenylase complex. Transcriptomic analysis reveals that the extent of suppression is dependent on the number of DND1-binding sites. This DND1-dependent mRNA destabilization is required for the survival of mouse PGCs and spermatogonial stem cells by suppressing apoptosis. The spectrum of target RNAs includes positive regulators of apoptosis and inflammation, and modulators of signalling pathways that regulate stem-cell pluripotency, including the TGFß superfamily, all of which are aberrantly elevated in DND1-deficient PGCs. We propose that the induction of the post-transcriptional suppressor DND1 synergizes with concurrent transcriptional changes to ensure precise developmental transitions during cellular differentiation and maintenance of the germ line.
Subject(s)
Multiprotein Complexes/metabolism , Neoplasm Proteins/metabolism , RNA Stability , RNA, Messenger/metabolism , Ribonucleases/metabolism , Spermatogonia/cytology , Stem Cells/cytology , Transcription Factors/metabolism , 3' Untranslated Regions/genetics , Animals , Apoptosis/genetics , Base Sequence , Binding Sites , Cell Differentiation/genetics , Gene Expression Profiling , Gene Silencing , Humans , Inflammation/genetics , Male , Mice , Multiprotein Complexes/chemistry , Neoplasm Proteins/deficiency , Nucleotide Motifs , Pluripotent Stem Cells/cytology , Protein Binding , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Ribonucleases/chemistry , Signal Transduction/genetics , Spermatogonia/metabolism , Stem Cells/metabolism , Transcription, Genetic/genetics , Transforming Growth Factor beta/geneticsABSTRACT
The study of protein-RNA interactions is critical for our understanding of cellular processes and regulatory circuits controlled by RNA binding proteins (RBPs). Recent next generation sequencing-based approaches significantly promoted our understanding of RNA biology and its importance for cell function. We present a streamlined protocol for Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP), a technique that allows for the characterization of RBP binding sites on target RNAs at nucleotide resolution and transcriptome-wide scale. PAR-CLIP involves irreversible UV-mediated crosslinking of RNAs labeled with photoreactive nucleosides to interacting proteins, followed by stringent purification steps and the conversion of crosslinked RNA into small RNA cDNA libraries compatible with next-generation sequencing. The defining hallmark of PAR-CLIP is a diagnostic mutation at the crosslinking site that is introduced into cDNA during the library preparation process. This feature allows for efficient computational removal of contaminating sequences derived from non-crosslinked fragments of abundant cellular RNAs. In the following, we present two different step-by-step procedures for PAR-CLIP, which differ in the small RNA cDNA library preparation procedure: (1) Standard library preparation involving gel size selections after each enzymatic manipulation, and (2) A modified PAR-CLIP procedure ("on-beads" PAR-CLIP), where most RNA manipulations including the necessary adapter ligation steps are performed on the immobilized RNP. This streamlined procedure reduces the protocol preparation time by three days compared to the standard workflow.
Subject(s)
Gene Library , High-Throughput Nucleotide Sequencing/methods , Immunoprecipitation/methods , RNA-Binding Proteins/genetics , RNA/chemistry , Sequence Analysis, RNA/methods , Thiouridine/metabolism , Antibodies/chemistry , Base Sequence , Binding Sites , Cell Line , Electrophoresis, Agar Gel/methods , Humans , Mutation , Phosphorus Radioisotopes , Protein Binding , RNA/genetics , RNA/metabolism , RNA-Binding Proteins/metabolism , Ribonucleases/chemistry , Software , Thiouridine/chemistry , Transcriptome , Ultraviolet Rays , Y-Box-Binding Protein 1/genetics , Y-Box-Binding Protein 1/metabolismABSTRACT
In this study, we examine the mechanism of nanopore-based DNA sequencing using a voltage bias across a graphene nanoribbon. Using density function theory and a nonequilibrium Green's function approach, we determine the transmission spectra and current profile for adenine, guanine, cytosine, thymine, and uracil as a function of bias voltage in an energy minimized configuration. Utilizing the transmission current, we provide a general methodology for the development of a three nanopore graphene-based device that can be used to distinguish between the various nucleobases for DNA/RNA sequencing. From our analysis, we deduce that it is possible to use different transverse currents across a multinanopore device to differentiate between nucleobases using various voltages of 0.5, 1.3, and 1.6 V. Overall, our goal is to improve nanopore design to further DNA/RNA nucleobase sequencing and biomolecule identification techniques.
