Identification of proteins and miRNAs that specifically bind an mRNA in vivo

Understanding regulation of an mRNA requires knowledge of its regulators. However, methods for reliable de-novo identification of proteins binding to a particular RNA are scarce and were thus far only successfully applied to abundant noncoding RNAs in cell culture. Here, we present vIPR, an RNA-protein crosslink, RNA pulldown, and shotgun proteomics approach to identify proteins bound to selected mRNAs in C. elegans. Applying vIPR to the germline-specific transcript gld-1 led to enrichment of known and novel interactors. By comparing enrichment upon gld-1 and lin-41 pulldown, we demonstrate that vIPR recovers both common and specific RNA-binding proteins, and we validate DAZ-1 as a specific gld-1 regulator. Finally, combining vIPR with small RNA sequencing, we recover known and biologically important transcript-specific miRNA interactions, and we identify miR-84 as a specific interactor of the gld-1 transcript. We envision that vIPR will provide a platform for investigating RNA in vivo regulation in diverse biological systems

Exact density profiles for fully asymmetric exclusion process with discrete-time dynamics

Exact density profiles in the steady state of the one-dimensional fully asymmetric simple exclusion process on semi-infinite chains are obtained in the case of forward-ordered sequential dynamics by taking the thermodynamic limit in our recent exact results for a finite chain with open boundaries. The corresponding results for sublattice parallel dynamics follow from the relationship obtained by Rajewsky and Schreckenberg [Physica A 245, 139 (1997)] and for parallel dynamics from the mapping found by Evans, Rajewsky and Speer [J. Stat. Phys. 95, 45 (1999)]. By comparing the asymptotic results appropriate for parallel update with those published in the latter paper, we correct some technical errors in the final results given there.Comment: About 10 pages and 3 figures, new references are added and a comparison is made with the results by de Gier and Nienhuis [Phys. Rev. E 59, 4899(1999)

Post-transcriptional regulation by 3' UTRs can be masked by regulatory elements in 5' UTRs

In mRNA sequences, 3' UTRs are thought to contain most elements that specifically regulate localization, turnover, and translation. Although high-throughput experiments indicate that many RNA-binding proteins (RBPs) also bind 5' UTRs, much less is known about specific post-transcriptional control exerted by 5' UTRs. GLD-1 is a conserved RBP and a translational repressor with essential roles in Caenorhabditis elegans germ cell development. Previously, we showed that GLD-1 binds highly conserved sites in both 3' and 5' UTRs. Here, by targeted single-copy insertion of transgenes, we systematically tested in vivo functionality of 5' and 3' UTR binding sites individually and in combination. Our data show that sites in 5' UTRs mediate specific and strong translational repression, independent of exact position. Intriguingly, we found that the functionality of 3' UTR sites can be masked by 5' UTR sites and vice versa. We conclude that it is important to study both UTRs simultaneously

Protocol for efficient CRISPR/Cas9/AAV-mediated homologous recombination in mouse hematopoietic stem and progenitor cells

Mutations that accumulate in self-renewing hematopoietic stem and progenitor cells (HSPCs) can cause severe blood disorders. To model such disorders in mice, we developed a CRISPR/Cas9/adeno-associated virus (AAV)-based system to knock in and repair genes by homologous recombination in mouse HSPCs. Here, we provide a step-by-step protocol to achieve high efficiency of gene knockin in mouse HSPCs, while maintaining engraftment capacity. This approach enables the functional study of hematopoietic disease mutations in vivo, without requiring germline mutagenesis

Application of the Density Matrix Renormalization Group Method to a Non-Equilibrium Problem

We apply the density matrix renormalization group (DMRG) method to a non-equilibrium problem: the asymmetric exclusion process in one dimension. We study the stationary state of the process to calculate the particle density profile (one-point function). We show that, even with a small number of retained bases, the DMRG calculation is in excellent agreement with the exact solution obtained by the matrix-product-ansatz approach.Comment: 8 pages, LaTeX (using jpsj.sty), 4 non-embedded figures, submitted to J. Phys. Soc. Jp

Exact solution of an exclusion process with three classes of particles and vacancies

We present an exact solution for an asymmetric exclusion process on a ring with three classes of particles and vacancies. Using a matrix product Ansatz, we find explicit expressions for the weights of the configurations in the stationary state. The solution involves tensor products of quadratic algebras.Comment: 18 pages, no figures, LaTe