A post-transcriptional regulatory switch in polypyrimidine tract-binding proteins reprograms alternative splicing in developing neurons

Abstract

Many metazoan gene transcripts exhibit neuron-specific splicing patterns, but the developmental control of these splicing events is poorly understood. We show that the splicing of a large group of exons is reprogrammed during neuronal development by a switch in expression between two highly similar polypyrimidine tract-binding proteins, PTB and nPTB (neural PTB). PTB is a well-studied regulator of alternative splicing, but nPTB is a closely related paralog whose functional relationship to PTB is unknown. In the brain, nPTB protein is specifically expressed in post-mitotic neurons, whereas PTB is restricted to neuronal precursor cells (NPC), glia, and other nonneuronal cells. Interestingly, nPTB mRNA transcripts are found in NPCs and other nonneuronal cells, but in these cells nPTB protein expression is repressed. This repression is due in part to PTB-induced alternative splicing of nPTB mRNA, leading to nonsense-mediated decay (NMD). However, we find that even properly spliced mRNA fails to express nPTB protein when PTB is present, indicating contributions from additional post-transcriptional mechanisms. The PTB-controlled repression of nPTB results in a mutually exclusive pattern of expression in the brain, where the loss of PTB in maturing neurons allows the synthesis of nPTB in these cells. To examine the consequences of this switch, we used splicing-sensitive microarrays to identify different sets of exons regulated by PTB, nPTB, or both proteins. During neuronal differentiation, the splicing of these exon sets is altered as predicted from the observed changes in PTB and nPTB expression. These data show that the post-transcriptional switch from PTB to nPTB controls a widespread alternative splicing program during neuronal development.[Keywords: Alternative splicing; neuronal development; nonsense-mediated decay; polypyrimidine tract-binding proteins; splicing microarray; ultraconserved element] Supplemental material is available at http://www.genesdev.org. Alternative pre-mRNA splicing is a common mechanism for diversifying genetic output in metazoan organisms (Black 2003;Matlin et al. 2005). Alternative choices in exons and splice sites can create substantial changes in the encoded protein and its activity. Changes in splicing can also affect downstream regulatory processes such as nonsense-mediated decay (NMD), and thus direct additional levels of post-transcriptional gene regulation (Lewis et al. 2003;Lejeune and Maquat 2005;Hughes 2006). Transcripts exhibiting multiple splicing patterns are especially prevalent in the mammalian nervous system, where alternative splicing affects important processes such as axon guidance, synaptogenesis, and the regulation of membrane physiology (Black and Grabowski 2003;Lipscombe 2005;. The choice of splicing pattern within a transcript is generally controlled by RNA-binding proteins that bind to the pre-mRNA to enhance or silence particular splicing events (Black 2003;Matlin et al. 2005). Some splicing regulators are expressed in a tissue-specific manner and have been sh...

Structure-function relationships of the polypyrimidine tract binding protein

“…Hence, neuronal nPTB shows significantly reduced repressor function [9]. Furthermore, a recent micro-array study showed that PTB and nPTB have a mutually exclusive expression and regulate different sets of alternative exons during neuron development [30]. It will be most interesting to see whether the functional differences between PTB and nPTB are reflected in the nPTB structure.…”

Introns in UTRs: Why we should stop ignoring them

“…Thus, as neurons differentiate and Ptbp1 levels decrease, the non 3UI-containing form of PTBP2 mRNA is produced. As a result, Ptbp2 protein levels increase, leading to a regulated change in alternative splicing of its many targets, many of which are involved in neuronal differentiation [75]. Some of these Ptbp2 splicing targets are themselves NMD substrates.…”

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“…Alternatively, two RBPs may bind simultaneously to a substrate and exert opposing influences on the spliceosome (Fu and Ares 2014). Antagonism at the level of expression also plays an important role in tuning RBP repertoire and activity, as transregulation of one RBP by others is common (Boutz et al 2007;Huelga et al 2012;Fu and Ares 2014).…”