TDP-43 is a critical RNA-binding factor associated with pre-mRNA splicing in mammals. Its expression is tightly autoregulated, with loss of this regulation implicated in human neuropathology. We demonstrate that TDP-43 overexpression in humans and mice activates a 39 untranslated region (UTR) intron, resulting in excision of the proximal polyA site (PAS) pA 1 . This activates a cryptic PAS that prevents TDP-43 expression through a nuclear retention mechanism. Superimposed on this process, overexpression of TDP-43 blocks recognition of pA 1 by competing with CstF-64 for PAS binding. Overall, we uncover complex interplay between transcription, splicing, and 39 end processing to effect autoregulation of TDP-43.

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.

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High mobility group A proteins (HMGA1 and HMGA2) are architectural factors involved in chromatin remodelling and regulation of gene expression. HMGA are highly expressed during embryogenesis and in cancer cells and are involved in development and cell differentiation as well as cancer formation and progression. These factors, by binding to DNA and interacting with other nuclear proteins, can organize macromolecular complexes involved in transcription, chromatin dynamics, RNA processing, and DNA repair. The identification of protein partners for HMGA has greatly contributed to our understanding of their multiple functions. He we report the identification of HMGA molecular partners using a gene fragment library in a phage display screening. Using an ORF-enriched cDNA library, we have isolated several HMGA1 interacting clones and for two of them, TBP associated factor 3 (TAF3) and chromatin assembly factor 1 p150/CAF-1, have demonstrated an in vivo association with HMGA1. The identification of these new partners suggests that HMGA can also influence general aspects of transcription and once more underlines their involvement in chromatin remodelling and dynamics.

Although the etiology of multiple sclerosis is not yet understood, it is accepted that its pathogenesis involves both autoimmune and neurodegenerative processes, in which the role of autoreactive T-cells has been elucidated. Instead, the contribution of humoral response is still unclear, even if the presence of intrathecal antibodies and B-cells follicle-like structures in meninges of patients has been demonstrated. Several myelin and non-myelin antigens have been identified, but none has been validated as humoral biomarker. In particular autoantibodies against myelin proteins have been found also in healthy individuals, whereas non-myelin antigens have been implicated in neurodegenerative phase of the disease.To provide further putative autoantigens of multiple sclerosis, we investigated the antigen specificity of immunoglobulins present both in sera and in cerebrospinal fluid of patients using phage display technology in a new improved format. A human brain cDNA phage display library was constructed and enriched for open-read-frame fragments. This library was selected against pooled and purified immunoglobulins from cerebrospinal fluid and sera of multiple sclerosis patients. The antigen library was also screened against an antibody scFv library obtained from RNA of B cells purified from the cerebrospinal fluid of two relapsing remitting patients. From all biopanning a complex of 14 antigens were identified; in particular, one of these antigens, corresponding to DDX24 protein, was present in all selections. The ability of more frequently isolated antigens to discriminate between sera from patients with multiple sclerosis or other neurological diseases was investigated. The more promising novel candidate autoantigens were DDX24 and TCERG1. Both are implicated in RNA modification and regulation which can be altered in neurodegenerative processes. Therefore, we propose that they could be a marker of a particular disease activity state.