An RNA-Seq Strategy to Detect the Complete Coding and Non-Coding Transcriptome Including Full-Length Imprinted Macro ncRNAs

Imprinted macro non-protein-coding (nc) RNAs are cis-repressor transcripts that silence multiple genes in at least three imprinted gene clusters in the mouse genome. Similar macro or long ncRNAs are abundant in the mammalian genome. Here we present the full coding and non-coding transcriptome of two mouse tissues: differentiated ES cells and fetal head using an optimized RNA-Seq strategy. The data produced is highly reproducible in different sequencing locations and is able to detect the full length of imprinted macro ncRNAs such as Airn and Kcnq1ot1, whose length ranges between 80–118 kb. Transcripts show a more uniform read coverage when RNA is fragmented with RNA hydrolysis compared with cDNA fragmentation by shearing. Irrespective of the fragmentation method, all coding and non-coding transcripts longer than 8 kb show a gradual loss of sequencing tags towards the 3′ end. Comparisons to published RNA-Seq datasets show that the strategy presented here is more efficient in detecting known functional imprinted macro ncRNAs and also indicate that standardization of RNA preparation protocols would increase the comparability of the transcriptome between different RNA-Seq datasets

A novel transgenic mouse strain expressing PKC beta II demonstrates expansion of B1 and marginal zone B cell populations

Protein kinase Cβ (PKCβ) expressed in mammalian cells as two splice variants, PKCβI and PKCβII, functions in the B cell receptor (BCR) signaling pathway and contributes to B cell development. We investigated the relative role of PKCβII in B cells by generating transgenic mice where expression of the transgene is directed to these cells using the Eµ promoter (Eµ-PKCβIItg). Our findings demonstrate that homozygous Eµ-PKCβIItg mice displayed a shift from IgD+IgMdim toward IgDdimIgM+ B cell populations in spleen, peritoneum and peripheral blood. Closer examination of these tissues revealed respective expansion of marginal zone (MZ)-like B cells (IgD+IgM+CD43negCD21+CD24+), increased populations of B-1 cells (B220+IgDdimIgM+CD43+CD24+CD5+), and higher numbers of immature B cells (IgDdimIgMdimCD21neg) at the expense of mature B cells (IgD+IgM+CD21+). Therefore, the overexpression of PKCβII, which is a phenotypic feature of chronic lymphocytic leukaemia cells, can skew B cell development in mice, most likely as a result of a regulatory influence on BCR signaling

Alternative Splicing and Nonsense-Mediated RNA Decay Contribute to the Regulation of SHOX Expression

The human SHOX gene is composed of seven exons and encodes a paired-related homeodomain transcription factor. SHOX mutations or deletions have been associated with different short stature syndromes implying a role in growth and bone formation. During development, SHOX is expressed in a highly specific spatiotemporal expression pattern, the underlying regulatory mechanisms of which remain largely unknown. We have analysed SHOX expression in diverse embryonic, fetal and adult human tissues and detected expression in many tissues that were not known to express SHOX before, e.g. distinct brain regions. By using RT-PCR and comparing the results with RNA-Seq data, we have identified four novel exons (exon 2a, 7-1, 7-2 and 7-3) contributing to different SHOX isoforms, and also established an expression profile for the emerging new SHOX isoforms. Interestingly, we found the exon 7 variants to be exclusively expressed in fetal neural tissues, which could argue for a specific role of these variants during brain development. A bioinformatical analysis of the three novel 3′UTR exons yielded insights into the putative role of the different 3′UTRs as targets for miRNA binding. Functional analysis revealed that inclusion of exon 2a leads to nonsense-mediated RNA decay altering SHOX expression in a tissue and time specific manner. In conclusion, SHOX expression is regulated by different mechanisms and alternative splicing coupled with nonsense-mediated RNA decay constitutes a further component that can be used to fine tune the SHOX expression level

Multi-State Regulation of the Dihydrogen Phosphate Binding Affinity to a Light- and Heat-Responsive Bis-Urea Receptor

A responsive bis-urea receptor can be switched between three isomers using light and heat as evidenced by H-1 NMR and UV-vis spectroscopy. Anion binding experiments H-1 NMR titrations, ESI-MS) reveal a high selectivity for dihydrogen phosphate. Importantly, a large difference in binding affinity to the interchangeable isomers is observed, which is further rationalized by DFT calculations. As a consequence, the amount of bound substrate can be controlled via photo- and thermal isomerization in a three-step process

Dynamic control over catalytic function using responsive bisthiourea catalysts

Two novel bisthiourea molecular motor-based catalysts were designed and their dynamic control of activity and stereoselectivity in the Henry reaction using heat and light as external stimuli was investigated. Enantioselective organocatalysis studies established that a basic functional group is not required within the catalyst core to effect efficient dual stereocontrol, illustrating novel stimuli responsive organocatalysts