Evidence that the nonsense-mediated mRNA decay pathway participates in X chromosome dosage compensation in mammals

Abstract

Current models of X chromosome dosage compensation are usually framed by reference to how regulation in transcriptional level elevates the gene expression of the active X chromosome. This framework, however, might be oversimplified because regulation of gene expression can also act at the post-transcriptional level. Here, after a genome-wide survey, we find that autosomal genes are more likely subject to nonsense-mediated mRNA decay (NMD) than X-linked genes. Furthermore, we demonstrate that after NMD inhibition, balanced gene expression between X chromosome and autosomes is corrupted such that the global mean X/autosome gene expression ratio is decreased by 10–15%. Our results identify NMD as a post-transcription-level regulatory mechanism that contributes to the observed fine-tuning of X chromosome dosage compensation in mammals.

Introduction

The nonsense-mediated mRNA decay (NMD) pathway is best known as an important surveillance mechanism that rids eukaryotic cells of aberrant transcripts coding for prematurely terminated, non-functional, or deleteriously truncated proteins [1], [2]. Premature translation termination codons (PTCs) induced by nonsense or frameshift mutations are recognized by the NMD machinery, after which enzymes involved in general mRNA decay are recruited to rapidly degrade the aberrant transcripts before they enter the translation process [3]. Alternative splicing is an important component of post-transcriptional regulation that greatly expands the diversity of expressed mRNA transcripts [3], [4]. Different mRNA isoforms are created by distinct linear arrangements of exons, and this process sometimes creates PTCs, causing the transcript to be targeted by NMD for rapid degradation. Examination of both individual and global alternative splicing events has revealed that some conserved and regulated alternative splicing introduces PTCs, and that the coupling of alternative splicing and NMD is pervasive [1], [5]. In light of these findings, the NMD pathway could be viewed both as a sensor for inappropriate polypeptide synthesis and as a regulator of gene expression.

In this study, we used EST alignments to identify maximally reliable sequences that are alternatively spliced, and used a bioinformatics approach to evaluate the prevalence of coupled alternative splicing-NMD. Generally, if translation terminates more than ∼50–55 nucleotides upstream of an exon–exon junction, then NMD will occur [5], [6]. Based on this rule, we conducted a large-scale analysis of reliable alternative isoforms of human and mouse transcriptomes to identify transcripts containing premature termination codons (PTCs) that are direct targets of NMD. We find that coupling of alternative splicing and NMD is pervasive [5], [6], and interestingly, the X chromosome has the least hitchhiking of apparent NMD targets of all the chromosomes examined, suggesting that mRNA degradation of X-linked genes via the NMD pathway might be weaker than that of autosomal genes. Whether this reflects a skewed activity of NMD-promoted RNA decay is however a matter yet to be resolved.

In higher eukaryotes with dimorphic sex chromosomes, such as humans and fruit flies, females have two X chromosomes, whereas males have only one. These organisms have evolved a mechanism that compensates not only for the different number of sex chromosomes between the two sexes but also for the differing X/autosome ratios. [7]. In mammals, one of the two X chromosomes in females is inactivated, and transcription from the active X chromosome in both males and females is subject to a roughly twofold increase to reach the level of the autosomes [7], [8], [9], as evidenced by microarray data demonstrating that the global mean X/autosome gene expression ratio is about 1 in all of the different somatic tissues [7]. The coordinated fine-tuning of transcription of a large number of X-linked genes and autosomal genes has fascinated generations of researchers. Despite the many efforts that have been made, however, a mechanistic explanation for equivalent expression between X chromosomes and autosomes in mammals remains elusive. Not surprisingly, most efforts have attempted to explain how transcription is elevated for the X chromosome [10]; however, gene expression is a complex process involving a number of interlinked steps that are subject to several different qualitative and quantitative controls [11]. Accordingly, it is important to ask if post-transcriptional regulation plays a role in fine-tuning X chromosome/autosome gene expression. If the skewed chromosomal distribution of apparent NMD targets could exert an observable influence on gene expression, we might then expect the X/autosome gene expression level to be reduced if the NMD pathway is inhibited. We therefore examined these issues by analyzing pair-wise gene expression profiles of six different human cell lines with or without NMD inhibition, either by emetine [12] or by depletion of a key component of the NMD system, URF1 [13]. We found that X chromosome dosage compensation is corrupted after NMD inhibition. NMD has played a role to minimize the difference in gene expression level between X chromosome and autosomes. Therefore our results constitute the first clear evidence that post-transcriptional regulation plays a role in dosage compensation in mammals.