Abstract
TDP-43 is a nuclear protein involved in many aspects of RNA metabolism. To ensure cellular viability, its expression levels within cells must be tightly regulated. We have previously demonstrated that TDP-43 autoregulation occurs through the activation of a normally silent intron in its 3′-UTR sequence that results in the use of alternative polyadenylation sites. In this work, we analyse which is the dominant event in autoregulation: the recognition of the splice sites of 3′-UTR intron 7 or the intrinsic quality of the alternative polyadenylation sites. A panel of minigene constructs was tested for autoregulation functionality, protein production and subcellular messenger RNA localization. Our data clearly indicate that constitutive spliceosome complex formation across intron 7 does not lead to high protein production but, on the contrary, to lower TDP-43 messenger RNA and protein levels. This is due to altered nucleocytoplasmic distribution of the RNA that is mostly retained in the nucleus and degraded. This study provides a novel in-depth characterization of how RNA binding proteins can autoregulate their own levels within cells, an essential regulatory process in maintaining cellular viability.
Highlights
Cell viability relies on the correct protein concentration levels within the various cellular compartments [1] and prevents the development of disease, especially at the neuronal level [2]
We have previously shown that a minimal transcript consisting in a portion of the TDP-43 30-UTR region fused to the Green Fluorescent Protein (GFP) reporter protein displays similar autoregulatory properties to those observed for endogenous TARDBP gene (Figure 1B, X7 construct) [14,15]
We have previously reported the central role played by the TDP-43 binding region (TDPBR) sequence and by the splice sites of intron 7
Summary
Cell viability relies on the correct protein concentration levels within the various cellular compartments [1] and prevents the development of disease, especially at the neuronal level [2]. There are several pathways used by the cell to achieve this, with protein expression regulation at the messenger RNA (mRNA) level being one of the most common due to its ability to act in an efficient and rapid manner This type of regulation is often seen in genes encoding for RNA binding proteins due to the fact that many of these are able to bind their own RNA. In the majority of these cases, the autoregulatory processes for these proteins are based on the selective triggering of a specific RNA degradation mechanism called nonsense-mediated decay (NMD) [13] Exceptions to this rule are represented by HuR [3] and possibly Tra2 [10] proteins where polyadenylation and translational mechanisms may be prevalent. Another notable exception to this NMD rule is represented by the mechanism described to occur for the nuclear factor TDP-43 [14,15]
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