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Plants, unlike animals, exhibit a very high degree of plasticity in their growth and development and employ diverse strategies to cope with the variations during diurnal cycles and stressful conditions. Plants and animals, despite their remarkable morphological and physiological differences, share many basic cellular processes and regulatory mechanisms. Alternative splicing (AS) is one such gene regulatory mechanism that modulates gene expression in multiple ways. It is now well established that AS is prevalent in all multicellular eukaryotes including plants and humans. Emerging evidence indicates that in plants, as in animals, transcription and splicing are coupled. Here, we reviewed recent evidence in support of co-transcriptional splicing in plants and highlighted similarities and differences between plants and humans. An unsettled question in the field of AS is the extent to which splice isoforms contribute to protein diversity. To take a critical look at this question, we presented a comprehensive summary of the current status of research in this area in both plants and humans, discussed limitations with the currently used approaches and suggested improvements to current methods and alternative approaches. We end with a discussion on the potential role of epigenetic modifications and chromatin state in splicing memory in plants primed with stresses.

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No abstract

The transfer of exosomes containing both genetic and protein materials is necessary for the control of the cancer cell microenvironment to promote tumor angiogenesis. The nature and function of proteins found in the exosomal cargo, and the mechanism of their action in membrane transport and related signaling events are not clearly understood. In this study, we demonstrate, in human lung cancer A549 cells, that the exosome release mechanism is closely linked to the multifaceted receptor sortilin (also called neurotensin receptor 3). Sortilin is already known to be important for cancer cell function. Here, we report for the first time its role in the assembly of a tyrosine kinase complex and subsequent exosome release. This new complex (termed the TES complex) is found in exosomes and results in the linkage of the two tyrosine kinase receptors TrkB (also known as NTRK2) and EGFR with sortilin. Using in vitro models, we demonstrate that this sortilin-containing complex exhibits a control on endothelial cells and angiogenesis activation through exosome transfer.

The mammalian oligosaccharyltransferase (OST) complex is composed of about eight subunits and mediates the N-glycosylation of nascent polypeptide chains entering the endoplasmic reticulum (ER). The conserved STT3 subunit of eukaryotic OST complexes has been identified as its catalytic centre, yet although many other subunits are equally well conserved their functions are unknown. We used RNA interference to investigate the function of ribophorin I, an ER-translocon-associated subunit of the OST complex previously shown to associate with newly synthesised membrane proteins. We show that ribophorin I dramatically enhances the N-glycosylation of selected membrane proteins and provide evidence that it is not essential for N-glycosylation per se. Parallel studies confirm that STT3 is essential for transferase activity of the complex, but reveal that the two mammalian isoforms are not functionally equivalent when modifying bona fide polypeptide substrates. We propose a new model for OST function where ribophorin I acts as a chaperone or escort to promote the N-glycosylation of selected substrates by the catalytic STT3 subunits.

BackgroundNormalization in real-time qRT-PCR is necessary to compensate for experimental variation. A popular normalization strategy employs reference gene(s), which may introduce additional variability into normalized expression levels due to innate variation (between tissues, individuals, etc). To minimize this innate variability, multiple reference genes are used. Current methods of selecting reference genes make an assumption of independence in their innate variation. This assumption is not always justified, which may lead to selecting a suboptimal set of reference genes.ResultsWe propose a robust approach for selecting optimal subset(s) of reference genes with the smallest variance of the corresponding normalizing factors. The normalizing factor variance estimates are based on the estimated unstructured covariance matrix of all available candidate reference genes, adjusting for all possible correlations. Robustness is achieved through bootstrapping all candidate reference gene data and obtaining the bootstrap upper confidence limits for the variances of the log-transformed normalizing factors. The selection of the reference gene subset is optimized with respect to one of the following criteria: (A) to minimize the variability of the normalizing factor; (B) to minimize the number of reference genes with acceptable upper limit on variability of the normalizing factor, (C) to minimize the average rank of the variance of the normalizing factor. The proposed approach evaluates all gene subsets of various sizes rather than ranking individual reference genes by their stability, as in the previous work. In two publicly available data sets and one new data set, our approach identified subset(s) of reference genes with smaller empirical variance of the normalizing factor than in subsets identified using previously published methods. A small simulation study indicated an advantage of the proposed approach in terms of sensitivity to identify the true optimal reference subset in the presence of even modest, especially negative correlation among the candidate reference genes.ConclusionsThe proposed approach performs comprehensive and robust evaluation of the variability of normalizing factors based on all possible subsets of candidate reference genes. The results of this evaluation provide flexibility to choose from important criteria for selecting the optimal subset(s) of reference genes, unless one subset meets all the criteria. This approach identifies gene subset(s) with smaller variability of normalizing factors than current standard approaches, particularly if there is some nontrivial innate correlation among the candidate genes.

Plants display exquisite control over gene expression to elicit appropriate responses under normal and stress conditions. Alternative splicing (AS) of pre-mRNAs, a process that generates two or more transcripts from multi-exon genes, adds another layer of regulation to fine-tune condition-specific gene expression in animals and plants. However, exactly how plants control splice isoform ratios and the timing of this regulation in response to environmental signals remains elusive. In mammals, recent evidence indicate that epigenetic and epitranscriptome changes, such as DNA methylation, chromatin modifications and RNA methylation, regulate RNA polymerase II processivity, co-transcriptional splicing, and stability and translation efficiency of splice isoforms. In plants, the role of epigenetic modifications in regulating transcription rate and mRNA abundance under stress is beginning to emerge. However, the mechanisms by which epigenetic and epitranscriptomic modifications regulate AS and translation efficiency require further research. Dynamic changes in the chromatin landscape in response to stress may provide a scaffold around which gene expression, AS and translation are orchestrated. Finally, we discuss CRISPR/Cas-based strategies for engineering chromatin architecture to manipulate AS patterns (or splice isoforms levels) to obtain insight into the epigenetic regulation of AS.