Functional Implications of Structural Predictions for Alternative Splice Proteins Expressed in Her2/neu–Induced Breast Cancers

Alternative splicing allows a single gene to generate multiple mRNA transcripts, which can be translated into functionally diverse proteins. However, experimentally determined structures of protein splice isoforms are rare, and homology modeling methods are poor at predicting atomic-level structural differences because of high sequence identity. Here we exploit the state-of-the-art structure prediction method I-TASSER to analyze the structural and functional consequences of alternative splicing of proteins differentially expressed in a breast cancer model. We first successfully benchmarked the I-TASSER pipeline for structure modeling of all seven pairs of protein splice isoforms, which are known to have experimentally solved structures. We then modeled three cancer-related variant pairs reported to have opposite functions. In each pair, we observed structural differences in regions where the presence or absence of a motif can directly influence the distinctive functions of the variants. Finally, we applied the method to five splice variants overexpressed in mouse Her2/neu mammary tumor: anxa6, calu, cdc42, ptbp1, and tax1bp3. Despite >75% sequence identity between the variants, structural differences were observed in biologically important regions of these protein pairs. These results demonstrate the feasibility of integrating proteomic analysis with structure-based conformational predictions of differentially expressed alternative splice variants in cancers and other conditions

Systematically Differentiating Functions for Alternatively Spliced Isoforms through Integrating RNA-seq Data

<div><p>Integrating large-scale functional genomic data has significantly accelerated our understanding of gene functions. However, no algorithm has been developed to differentiate functions for isoforms of the same gene using high-throughput genomic data. This is because standard supervised learning requires ‘ground-truth’ functional annotations, which are lacking at the isoform level. To address this challenge, we developed a generic framework that interrogates public RNA-seq data at the transcript level to differentiate functions for alternatively spliced isoforms. For a specific function, our algorithm identifies the ‘responsible’ isoform(s) of a gene and generates classifying models at the isoform level instead of at the gene level. Through cross-validation, we demonstrated that our algorithm is effective in assigning functions to genes, especially the ones with multiple isoforms, and robust to gene expression levels and removal of homologous gene pairs. We identified genes in the mouse whose isoforms are predicted to have disparate functionalities and experimentally validated the ‘responsible’ isoforms using data from mammary tissue. With protein structure modeling and experimental evidence, we further validated the predicted isoform functional differences for the genes <i>Cdkn2a</i> and <i>Anxa6</i>. Our generic framework is the first to predict and differentiate functions for alternatively spliced isoforms, instead of genes, using genomic data. It is extendable to any base machine learner and other species with alternatively spliced isoforms, and shifts the current gene-centered function prediction to isoform-level predictions.</p></div

Overview of the computational approach for predicting functions for alternatively spliced isoforms.

<p>We collected RNA-seq data from the sequence read archive (SRA) database and estimated isoform-level expression values using state-of-the-art software <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003314#pcbi.1003314-Trapnell1" target="_blank">[32]</a>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003314#pcbi.1003314-Trapnell2" target="_blank">[34]</a>. We then generated a gene-level gold standard using Gene Ontology (GO) annotations. For each biological function, this gold standard contains positive genes (annotated to the function under investigation) and negative genes (other genes). Our study contains two major parts: cross-validation for performance estimation and bootstrap bagging for generating final predictions as well as performance evaluation. For cross-validation, we partitioned the examples into a training set for model development and a test set for model validation. For generating final predictions for all isoforms, we sampled with replacement to construct a training set, and then used this training set to construct models to assign prediction probabilities to the out-of-bag set. The final predictions for all isoforms were made by calculating the median prediction values of all out-of-bag sets. For each training set, a model was derived from the RNA-seq data to delineate the positives and the negatives. This model was used to classify the training set and update the labels of the isoforms of the positive genes, under the criterion that at least one isoform of a positive gene must remain positive. This new assignment is then used to construct the model in the next iteration. This process is iterated until the assignment of positive isoforms no longer changes, and then the final model was used to assign a prediction value to the test or the out-of-the-bag set. Bootstrap was done for 30 iterations and the median value for each out-of-the-bag isoform was taken as the final prediction value. The predictive performance of our model was assessed through three approaches: (1) cross-validation of gene-level predictive performances, focusing on comparison between single-isoform genes and multiple-isoform genes, (2) literature validation and (3) experimental validation of top predictions using proteomic data.</p

Robust performance of our algorithm to predicting functions using RNA-seq data.

<p>We carried out five-fold cross validation to test the performance of our algorithm. For each function, the prediction value for each gene is assigned the maximum prediction value of all of its isoforms, under the assumption that at least one of its isoforms should carry out the function. Because the number of known genes of each GO term systematically affects the prediction performance, we group these terms into 5 groups according to their GO term sizes. (A)–(D) shows the distribution (10, 25, 50, 75, 90%) of the AUCs, the AUPRCs, the precisions at 1% recall and the precisions at 10% recall, respectively.</p

Prediction precision between single-isoform genes (green) with multi-isoform gene (blue).

<p>Two-fold cross-validation was carried out to ensure that enough examples are included in both the single-isoform group and the multi-isoform group. The negatives were randomly selected to ensure that the ratios of positive to negative genes for the multi-isoform group and the single isoform group are the same for each GO term, so that the baseline precision for each GO term is equal for the two groups. GO terms were grouped according to the number of genes in the test set. Each dot represents the precision value of an individual GO term. A. Precision at one percent recall. B. Precision at ten percent recall.</p

Predicted functions for isoforms of CDKN2a and their predicted protein structures. A

<p>. Gene model for NM_001040654.1 and NM_009877.2. <b>B</b>. Predicted functions for NM_001040654.1 and NM_009877.2. <b>C</b>. The computationally modeled structure of NM_001040654.1 is characterized by five ankryin repeats. <b>D</b>. The modeled structure of NM_009877.2 has a CDKN2a N-terminus domain.</p

Examples for predicted functional isoforms that are validated using proteomic data.

<p>Examples for predicted functional isoforms that are validated using proteomic data.</p

Prediction performance comparison of single-isoform genes (green) and multi-isoform gene (blue) based on AUC (upper panel) and AUPRC (lower panel).

<p>We separately evaluated its prediction performance for single-isoform genes and multiple-isoform genes. Two-fold cross-validation was carried out to ensure enough examples in both groups. To ensure comparability, the negatives were randomly selected to ensure that the ratios of positive to negative genes for the multi-isoform group and the single isoform group are the same for each GO term. GO terms were grouped according to the number of genes in the test set. Shown in the box-plot are the AUC (<b>A</b>) and AUPRC (<b>B</b>) at 10, 25, 50, 75 and 90 percentile, respectively.</p

Functional Networks of Highest-Connected Splice Isoforms: From The Chromosome 17 Human Proteome Project

Alternative splicing allows a single gene to produce multiple transcript-level splice isoforms from which the translated proteins may show differences in their expression and function. Identifying the major functional or canonical isoform is important for understanding gene and protein functions. Identification and characterization of splice isoforms is a stated goal of the HUPO Human Proteome Project and of neXtProt. Multiple efforts have catalogued splice isoforms as “dominant”, “principal”, or “major” isoforms based on expression or evolutionary traits. In contrast, we recently proposed highest connected isoforms (HCIs) as a new class of canonical isoforms that have the strongest interactions in a functional network and revealed their significantly higher (differential) transcript-level expression compared to nonhighest connected isoforms (NCIs) regardless of tissues/cell lines in the mouse. HCIs and their expression behavior in the human remain unexplored. Here we identified HCIs for 6157 multi-isoform genes using a human isoform network that we constructed by integrating a large compendium of heterogeneous genomic data. We present examples for pairs of transcript isoforms of <i>ABCC3, RBM34</i>, <i>ERBB2</i>, and <i>ANXA7</i>. We found that functional networks of isoforms of the same gene can show large differences. Interestingly, differential expression between HCIs and NCIs was also observed in the human on an independent set of 940 RNA-seq samples across multiple tissues, including heart, kidney, and liver. Using proteomic data from normal human retina and placenta, we showed that HCIs are a promising indicator of expressed protein isoforms exemplified by <i>NUDFB6</i> and <i>M6PR</i>. Furthermore, we found that a significant percentage (20%, <i>p</i> = 0.0003) of human and mouse HCIs are homologues, suggesting their conservation between species. Our identified HCIs expand the repertoire of canonical isoforms and are expected to facilitate studying main protein products, understanding gene regulation, and possibly evolution. The network is available through our web server as a rich resource for investigating isoform functional relationships (http://guanlab.ccmb.med.umich.edu/hisonet). All MS/MS data were available at ProteomeXchange Web site (http://www.proteomexchange.org) through their identifiers (retina: PXD001242, placenta: PXD000754)

Performance comparison of different formulations of the SVM-MIL algorithm in predicting isoform functions.

<p>A. The histogram shows the score distribution of the instances in the positive bags and the negative bags in the training set. Different threshold choices in mi-SVM are based on the distribution of scores of negative genes. The first threshold is equal to the mode of distribution of scores from negative instances in the training set. The second threshold is equal to the 75% percentile of scores of the negative instances in the training set. The third threshold is equal to the maximum score of negative instances in the training set. B. This panel illustrates how different thresholds and formulations can divide the isoforms in a positive bag into positive, negative and neutral classes. Three thresholds in mi-SVM represent different degrees of strictness for assigning labels. The first threshold is the least strict, which assigns most of the isoforms from positive genes as positive, whereas the third threshold is the strictest, which in general leaves only one positive instance in every positive bag. For the MI-SVM formulation, only one isoform per positive gene is assigned as positive, and other isoforms are dropped (<i>i.e.</i> neutral class). C. Performance comparison of three different threshold choices for the mi-SVM formulation, the MI-SVM formulation and the MI-SVM formulation with random witness selection. This plot shows that the mi-SVM formulation with threshold-2 performs best in terms of AUC.</p