TDP-43 Binds Heterogeneous Nuclear Ribonucleoprotein A/B through Its C-terminal Tail

Abstract

TDP-43 is a highly conserved nuclear factor of yet unknown function that binds to ug-repeated sequences and is responsible for cystic fibrosis transmembrane conductance regulator exon 9 splicing inhibition. We have analyzed TDP-43 interactions with other splicing factors and identified the critical regions for the protein/protein recognition events that determine this biological function. We show here that the C-terminal region of TDP-43 is capable of binding directly to several proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) family with well known splicing inhibitory activity, in particular, hnRNP A2/B1 and hnRNP A1. Mutational analysis showed that TDP-43 proteins lacking the C-terminal region could not inhibit splicing probably because they were unable to form the hnRNP-rich complex involved in splicing inhibition. Finally, through splicing complex analysis, we show that splicing inhibition mediated by TDP-43 occurs at the earliest stages of spliceosomal assembly.In its most basic form, the splicing process has the task of removing from the primary RNA transcript all of those sequences (introns) that will not be present in the mature mRNA (1-3). Alternative splicing, i.e. the inclusion/exclusion of selected exonic sequences in particular tissues or developmental stages, has been heavily exploited by evolution to generate multiple mRNA transcripts from the same pre-mRNA sequence (4 -6). However, all of this flexibility also means that the splicing process is prone to mistakes following even minor changes (7), and alterations of splicing are being increasingly reported as the underlying cause of many genetic diseases (8 -12).At the molecular level, the removal of introns and the joining of exons are catalyzed by the spliceosome, which contains several hundred different proteins in addition to the five spliceosomal small nuclear RNAs (13,14). This complex arrangement of factors has two functions: first, to define the exact boundaries of an exon; and second, to catalyze the cut-and-paste generation of the mature mRNA. However, many external factors can also contribute to its workings, such as RNA secondary structure (15), transcription rates (16), the presence of splicing enhancer and silencer elements (17, 18), and even external stimuli (19,20). It is the combinatorial effect of all of these factors that will decide when, where, and to what degree a specific sequence will be included or not in the mature mRNA (17). Recently, the finding that at least 5% of all human alternative exons are derived from the highly repeated dimeric retrotransposons Alu elements has focused a lot of attention on the potential splicing modulatory ability of repeated nucleotide sequences (21).In previous works, we have focused our attention on clarifying the pathological role played by (ug)m-repeated sequences near the 3Ј-splice site of cystic fibrosis transmembrane conductance regulator (CFTR) 3 exon 9 (22-24), as they have been known to promote skipping and to correlate well with disease penetrance (25). In particular,...

A “Two-hit” Hypothesis for Inclusion Formation by Carboxyl-terminal Fragments of TDP-43 Protein Linked to RNA Depletion and Impaired Microtubule-dependent Transport

“…Studies of TDP-43 complexes with protein and RNA are beginning to provide a clearer understanding of TDP-43 as a component of large RNP complexes (9,41,42). Our observations that CTF transport are dependent on interactions of TDP-43 with RNA and on dynein-mediated MT transport are consistent with its classification as a shuttling RNP protein (12).…”

“…Both RRMs retain nucleic acid binding properties, yet only RRM1 appears essential for RNA splicing (8). The glycine-rich domain is proposed to interact with other hnRNPs to synergistically affect alternative splicing of specific RNA transcripts (9,10). A common feature of shuttling hnRNPs involved in RNA splicing is that their nuclear export is coupled to the export and maturation of mRNA in distinct ribonucleoprotein (RNP) complexes (11,12).…”

TDP-43 Is a Developmentally Regulated Protein Essential for Early Embryonic Development

“…In addition, TDP-43 has been implicated in regulation of mRNA biogenesis (9) and shown to be localized to sites of mRNA transcription and processing in neurons (10). As the glycine-rich domain of TDP-43 has been shown to mediate interactions with other heterogeneous nuclear ribonucleoprotein proteins, the low homology of this particular domain may afford a multitude of interactions that allows for diverse biological functions (11).…”

A novel SDS-stable dimer of a heterogeneous nuclear ribonucleoprotein at presynaptic terminals of squid neurons

“…This includes the hnRNPA/B proteins, which are prone to form these structures, especially when exacerbated by disease mutations (Kim et al 2013). Furthermore, hnRNPA2/B1 and hnRNP A1 are capable of binding with TAR DNA binding protein-43 (TDP-43) (Buratti et al, 2005), an important hnRNP identified in inclusion bodies of patients with amyotrophic lateral sclerosis and frontotemperal lobar degeneration (Winton et al, 2008). Note that TDP-43 is composed of an N-terminal region with two RNA recognition motifs and a glycine-rich carboxy terminal domain similar to the squid hnRNPA/B-like proteins, although it does not share primary sequence homology with any that we have identified.…”