Comparison of basic peptides- and lipid-based strategies for the delivery of splice correcting oligonucleotides

Expression of alternatively spliced mRNA variants at specific stages of development or in specific cells and tissues contributes to the functional diversity of the human genome. Aberrations in alternative splicing were found as a cause or a contributing factor to the development, progression, or maintenance of numerous diseases. The use of antisense oligonucleotides (ON) to modify aberrant expression patterns of alternatively spliced mRNAs is a novel means of potentially controlling such diseases. Oligonucleotides can be designed to repair genetic mutations, to modify genomic sequences in order to compensate for gene deletions, or to modify RNA processing in order to improve the effects of the underlying gene mutation. Steric block ON approach have proven to be effective in experimental model for various diseases. Here, we describe our experience in investigating two strategies for ON delivery: ON conjugation with basic peptides and lipid-based particulate system (lipoplex). Basic peptides or Cell Penetrating Peptides (CPP) such as the TAT-derived peptide appear to circumvent many problems associated with ON and drug delivery. This strategy may represent the next paradigm in our ability to modulate cell function and offers a unique avenue for the treatment of disease. Lipoplexes result from the intimate interaction of ON with cationic lipids leading to ON carrying particles able to be taken up by cells and to release ON in the cytoplasm. We have used as an experimental model the correction of a splicing alteration of the mutated β-globin intron causing thalassemia. Data on cell penetration and efficacy of correction of specific steric block ON delivered either by basic peptides or lipoplex are described. A comparison of the properties of both delivery systems is made respective to the use of this new class of therapeutic molecules.     

A three-dimensional model of the U1 small nuclear ribonucleoprotein particle

Most of the pre-mRNAs in the eukaryotic cell are comprised of protein-coding exons and non-protein-coding introns. The introns are removed and the exons are ligated together, or spliced, by a large, macromolecular complex known as the spliceosome. This RNA-protein assembly is made up of five uridine-rich small nuclear RNAs (U1-, U2-, U4-, U5- and U6-snRNA) as well over 300 proteins, which form small nuclear ribonucleoprotein particles (snRNPs). Initial recognition of the 5′ exon/intron splice site is mediated by the U1 snRNP, which is composed of the U1 snRNA as well as at least ten proteins. By combining structural informatics tools with the available biochemical and crystallographic data, we attempted to simulate a complete, three dimensional U1 snRNP from the silk moth, Bombyx mori. Comparison of our model with empirically derived crystal structures and electron micrographs pinpoints both the strengths and weaknesses in the in silico determination of macromolecular complexes. One of the most striking differences between our model and experimentally generated structures is in the positioning of the U1 snRNA stem-loops. This highlights the continuing difficulties in generating reliable, complex RNA structures; however, three-dimensional modeling of individual protein subunits by threading provided models of biological significance and the use of both automated and manual docking strategies generated a complex that closely reflects the assembly found in nature. Yet, without utilizing experimentally-derived contacts to select the most likely docking scenario, ab initio docking would fall short of providing a reliable model. Our work shows that the combination of experimental data with structural informatics tools can result in generation of near-native macromolecular complexes.     

Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions

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Arabidopsis consensus intron sequences

We have analysed 998 Arabidopsis intron sequences in the EMBL database. All Arabidopsis introns to adhere to the :GU...AG: rule with the exception of 1% of introns with :GC at their 5 ends. Virtually all of the introns contained a putative branchpoint sequence (YUNAN) 18 to 60 nt upstream of the 3 splice site. Although a polypyrimidine tract was much less apparent than in vertebrate introns, the most common nucleotide in the region upstream of the 3 splice site was uridine. Consensus sequences for 5 and 3 splice sites and branchpoint sequences for Arabidopsis introns are presented.     

Analysis of splice donor and acceptor site function in a transposable gene trap derived from the maize element Activator

Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3 Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.     

Splicing and alternative splicing in rice and humans

Rice is a monocot gramineous crop, and one of the most important staple foods. Rice is considered a model species for most gramineous crops. Extensive research on rice has provided critical guidance for other crops, such as maize and wheat. In recent years, climate change and exacerbated soil degradation have resulted in a variety of abiotic stresses, such as greenhouse effects, lower temperatures, drought, floods, soil salinization and heavy metal pollution. As such, there is an extremely high demand for additional research, in order to address these negative factors. Studies have shown that the alternative splicing of many genes in rice is affected by stress conditions, suggesting that manipulation of the alternative splicing of specific genes may be an effective approach for rice to adapt to abiotic stress. With the advancement of microarrays, and more recently, next generation sequencing technology, several studies have shown that more than half of the genes in the rice genome undergo alternative splicing. This mini-review summarizes the latest progress in the research of splicing and alternative splicing in rice, compared to splicing in humans. Furthermore, we discuss how additional studies may change the landscape of investigation of rice functional genomics and genetically improved rice. [BMB Reports 2013; 46(9): 439-447]     

PRPF6 promotes androgen receptor/androgen receptor-variant 7 actions in castration-resistant prostate cancer cells

Androgen receptor (AR) and its variants play vital roles in development and progression of prostate cancer. To clarify the mechanisms involved in the enhancement of their actions would be crucial for understanding the process in prostate cancer and castration-resistant prostate cancer transformation. Here, we provided the evidence to show that pre-mRNA processing factor 6 (PRPF6) acts as a key regulator for action of both AR full length (AR-FL) and AR variant 7 (AR-V7), thereby participating in the enhancement of AR-FL and AR-V7-induced transactivation in prostate cancer. In addition, PRPF6 is recruited to cis-regulatory elements in AR target genes and associates with JMJD1A to enhance AR-induced transactivation. PRPF6 also promotes expression of AR-FL and AR-V7. Moreover, PRPF6 depletion reduces tumor growth in prostate cancer-derived cell lines and results in significant suppression of xenograft tumors even under castration condition in mouse model. Furthermore, PRPF6 is obviously highly expressed in human prostate cancer samples. Collectively, our results suggest PRPF6 is involved in enhancement of oncogenic AR signaling, which support a previously unknown role of PRPF6 during progression of prostate cancer and castration-resistant prostate cancers.