Dissecting the target specificity of RNase H recruiting oligonucleotides using massively parallel reporter analysis of short RNA motifs.

Processing and post-transcriptional regulation of RNA often depend on binding of regulatory molecules to short motifs in RNA. The effects of such interactions are difficult to study, because most regulatory molecules recognize partially degenerate RNA motifs, embedded in a sequence context specific for each RNA. Here, we describe Library Sequencing (LibSeq), an accurate massively parallel reporter method for completely characterizing the regulatory potential of thousands of short RNA sequences in a specific context. By sequencing cDNA derived from a plasmid library expressing identical reporter genes except for a degenerate 7mer subsequence in the 3'UTR, the regulatory effects of each 7mer can be determined. We show that LibSeq identifies regulatory motifs used by RNA-binding proteins and microRNAs. We furthermore apply the method to cells transfected with RNase H recruiting oligonucleotides to obtain quantitative information for >15000 potential target sequences in parallel. These comprehensive datasets provide insights into the specificity requirements of RNase H and allow a specificity measure to be calculated for each tested oligonucleotide. Moreover, we show that inclusion of chemical modifications in the central part of an RNase H recruiting oligonucleotide can increase its sequence-specificity.

Pharmaco-miR: linking microRNAs and drug effects.

MicroRNAs (miRNAs) are short regulatory RNAs that down-regulate gene expression. They are essential for cell homeostasis and active in many disease states. A major discovery is the ability of miRNAs to determine the efficacy of drugs, which has given rise to the field of 'miRNA pharmacogenomics' through 'Pharmaco-miRs'. miRNAs play a significant role in pharmacogenomics by down-regulating genes that are important for drug function. These interactions can be described as triplet sets consisting of a miRNA, a target gene and a drug associated with the gene. We have developed a web server which links miRNA expression and drug function by combining data on miRNA targeting and protein-drug interactions. miRNA targeting information derive from both experimental data and computational predictions, and protein-drug interactions are annotated by the Pharmacogenomics Knowledge base (PharmGKB). Pharmaco-miR's input consists of miRNAs, genes and/or drug names and the output consists of miRNA pharmacogenomic sets or a list of unique associated miRNAs, genes and drugs. We have furthermore built a database, named Pharmaco-miR Verified Sets (VerSe), which contains miRNA pharmacogenomic data manually curated from the literature, can be searched and downloaded via Pharmaco-miR and informs on trends and generalities published in the field. Overall, we present examples of how Pharmaco-miR provides possible explanations for previously published observations, including how the cisplatin and 5-fluorouracil resistance induced by miR-148a may be caused by miR-148a targeting of the gene KIT. The information is available at www.Pharmaco-miR.org.

Origin of introns by 'intronization' of exonic sequences.

The mechanisms of spliceosomal intron creation have proved elusive. Here we describe a new mechanism: the recruitment of internal exonic sequences ('intronization') in Caenorhabditis species. The numbers of intronization events and introns gained by other mechanisms are similar, suggesting that intronization significantly contributes to recent intron creation in nematodes. Intronization is more common than the reverse process, loss of splicing of retained introns. Finally, these findings link alternative splicing with modern intron creation.

Pharmacogenomics genes show varying perceptibility to microRNA regulation.

OBJECTIVE: The aim of pharmacogenomics is to identify individual differences in genome and transcriptome composition and their effect on drug efficacy. MicroRNAs (miRNAs) are short noncoding RNAs that negatively regulate expression of the majority of animal genes, including many genes involved in drug efficacy. Consequently, differences in the miRNA expression among individuals could be an important factor contributing to differential drug response. Pharmacogenomics genes can be divided into drug target genes termed as pharmacodynamics genes (PD) and genes involved in drug transport and metabolism termed as pharmacokinetics genes (PK). To clarify the regulatory potential of miRNAs in pharmacogenomics, we have examined the potential regulation by miRNAs of PK and PD genes. METHODS: We identified PK and PD genes as annotated by the Pharmacogenomics Knowledge Base and examined miRNA targeting of genes in the two groups according to several miRNA target prediction databases. We furthermore studied how differences between the two groups are reflected in the gene structure and across gene families. Lastly, we studied changes in expression levels of PK versus PD genes in cells depleted for miRNAs by shRNA-mediated knockdown of the miRNA-processing enzyme Dicer. RESULTS: Our analysis identify a striking difference in the level of miRNA regulation between PK and PD genes, with the former having less than half predicted conserved miRNA binding sites compared with the latter. Importantly, this finding is reflected in a highly significant difference in the shift in expression levels of PD versus PK genes after depletion of miRNAs. CONCLUSION: Our study emphasizes an intrinsic difference between PK and PD genes and helps clarify the role of miRNAs in pharmacogenomics.

Functional and evolutionary analysis of alternatively spliced genes is consistent with an early eukaryotic origin of alternative splicing.

BACKGROUND: Alternative splicing has been reported in various eukaryotic groups including plants, apicomplexans, diatoms, amoebae, animals and fungi. However, whether widespread alternative splicing has evolved independently in the different eukaryotic groups or was inherited from their last common ancestor, and may therefore predate multicellularity, is still unknown. To better understand the origin and evolution of alternative splicing and its usage in diverse organisms, we studied alternative splicing in 12 eukaryotic species, comparing rates of alternative splicing across genes of different functional classes, cellular locations, intron/exon structures and evolutionary origins. RESULTS: For each species, we find that genes from most functional categories are alternatively spliced. Ancient genes (shared between animals, fungi and plants) show high levels of alternative splicing. Genes with products expressed in the nucleus or plasma membrane are generally more alternatively spliced while those expressed in extracellular location show less alternative splicing. We find a clear correspondence between incidence of alternative splicing and intron number per gene both within and between genomes. In general, we find several similarities in patterns of alternative splicing across these diverse eukaryotes. CONCLUSION: Along with previous studies indicating intron-rich genes with weak intron boundary consensus and complex spliceosomes in ancestral organisms, our results suggest that at least a simple form of alternative splicing may already have been present in the unicellular ancestor of plants, fungi and animals. A role for alternative splicing in the evolution of multicellularity then would largely have arisen by co-opting the preexisting process.

MicroRNA pharmacogenomics: post-transcriptional regulation of drug response.

The field of pharmacogenomics aims to predict which drugs will be most effective and safe for a particular individual based on their genome sequence or expression profile, thereby allowing personalized treatment. The bulk of pharmacogenomic research has focused on the role of single nucleotide polymorphisms, copy number variations or differences in gene expression levels of drug metabolizing or transporting genes and drug targets. In this review paper, we focus instead on microRNAs (miRNAs): small noncoding RNAs, prevalent in metazoans, that negatively regulate gene expression in many cellular processes. We discuss how miRNAs, by regulating the expression of pharmacogenomic-related genes, can play a pivotal role in drug efficacy and toxicity and have potential clinical implications for personalized medicine.

Evolution of alternative splicing regulation: changes in predicted exonic splicing regulators are not associated with changes in alternative splicing levels in primates.

Alternative splicing is tightly regulated in a spatio-temporal and quantitative manner. This regulation is achieved by a complex interplay between spliceosomal (trans) factors that bind to different sequence (cis) elements. cis-elements reside in both introns and exons and may either enhance or silence splicing. Differential combinations of cis-elements allows for a huge diversity of overall splicing signals, together comprising a complex 'splicing code'. Many cis-elements have been identified, and their effects on exon inclusion levels demonstrated in reporter systems. However, the impact of interspecific differences in these elements on the evolution of alternative splicing levels has not yet been investigated at genomic level. Here we study the effect of interspecific differences in predicted exonic splicing regulators (ESRs) on exon inclusion levels in human and chimpanzee. For this purpose, we compiled and studied comprehensive datasets of predicted ESRs, identified by several computational and experimental approaches, as well as microarray data for changes in alternative splicing levels between human and chimpanzee. Surprisingly, we found no association between changes in predicted ESRs and changes in alternative splicing levels. This observation holds across different ESR exon positions, exon lengths, and 5' splice site strengths. We suggest that this lack of association is mainly due to the great importance of context for ESR functionality: many ESR-like motifs in primates may have little or no effect on splicing, and thus interspecific changes at short-time scales may primarily occur in these effectively neutral ESRs. These results underscore the difficulties of using current computational ESR prediction algorithms to identify truly functionally important motifs, and provide a cautionary tale for studies of the effect of SNPs on splicing in human disease.

High qualitative and quantitative conservation of alternative splicing in Caenorhabditis elegans and Caenorhabditis briggsae.

Alternative splicing (AS) is an important contributor to proteome diversity and is regarded as an explanatory factor for the relatively low number of human genes compared with less complex animals. To assess the evolutionary conservation of AS and its developmental regulation, we have investigated the qualitative and quantitative expression of 21 orthologous alternative splice events through the development of 2 nematode species separated by 85-110 Myr of evolutionary time. We demonstrate that most of these alternative splice events present in Caenorhabditis elegans are conserved in Caenorhabditis briggsae. Moreover, we find that relative isoform expression levels vary significantly during development for 78% of the AS events and that this quantitative variation is highly conserved between the 2 species. Our results suggest that AS is generally tightly regulated through development and that the regulatory mechanisms controlling AS are to a large extent conserved during the evolution of Caenorhabditis. This strong conservation indicates that both major and minor splice forms have important functional roles and that the relative quantities in which they are expressed are crucial. Our results therefore suggest that the quantitative regulation of isoform expression levels is an intrinsic part of most AS events. Moreover, our results indicate that AS contributes little to transcript variation in Caenorhabditis genes and that gene duplication may be the major evolutionary mechanism for the origin of novel transcripts in these 2 species.