Laura De Conti

TDP‐43 regulates its mRNA levels through a negative feedback loop

TAR DNA‐binding protein (TDP‐43) is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in RNA processing, whose abnormal cellular distribution and post‐translational modification are key markers of certain neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We generated human cell lines expressing tagged forms of wild‐type and mutant TDP‐43 and observed that TDP‐43 controls its own expression through a negative feedback loop. The RNA‐binding properties of TDP‐43 are essential for the autoregulatory activity through binding to 3′ UTR sequences in its own mRNA. Our analysis indicated that the C‐terminal region of TDP‐43, which mediates TDP‐43–hnRNP interactions, is also required for self‐regulation. TDP‐43 binding to its 3′ UTR does not significantly change the pre‐mRNA splicing pattern but promotes RNA instability. Moreover, blocking exosome‐mediated degradation partially recovers TDP‐43 levels. Our findings demonstrate that cellular TDP‐43 levels are under tight control and it is likely that disease‐associated TDP‐43 aggregates disrupt TDP‐43 self‐regulation, thus contributing to pathogenesis.

Nuclear factor TDP-43 can affect selected microRNA levels.

TDP‐43 has recently been described as the major component of the inclusions found in the brain of patients with a variety of neurodegenerative diseases, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP‐43 is a ubiquitous protein whose specific functions are probably crucial to establishing its pathogenic role. Apart from its involvement in transcription, splicing and mRNA stability, TDP‐43 has also been described as a Drosha‐associated protein. However, our knowledge of the role of TDP‐43 in the microRNA (miRNA) synthesis pathway is limited to the association mentioned above. Here we report for the first time which changes occur in the total miRNA population following TDP‐43 knockdown in culture cells. In particular, we have observed that let‐7b and miR‐663 expression levels are down‐ and upregulated, respectively. Interestingly, both miRNAs are capable of binding directly to TDP‐43 in different positions: within the miRNA sequence itself (let‐7b) or in the hairpin precursor (miR‐663). Using microarray data and real‐time PCR we have also identified several candidate transcripts whose expression levels are selectively affected by these TDP‐43–miRNA interactions.

Exon and intron definition in pre-mRNA splicing

One of the fundamental issues in RNA splicing research is represented by understanding how the spliceosome can successfully define exons and introns in a huge variety of pre‐mRNA molecules with nucleotide‐precision. Since its first description, researchers in this field have identified and characterized many fundamental elements and players capable of affecting the splicing process, both in a negative and positive manner. Indeed, it can be argued that today we know a great deal about the forces that make an exon, an exon and an intron, an intron. As will be discussed in this review, these decisions are a result of a complex combinatorial control resulting from many different factors/influences. Most importantly, these influences act across several levels of complexity starting from the relatively simple interaction between two consensus 5′ and 3′ splice sites to much more complex factors: such as the interplay between silencer or enhancer sequences, transcriptional processivity, genomic milieu, nucleosome positioning, and histone modifications at the chromatin level. Depending on local contexts, all these factors will act either antagonistically or synergistically to decide the exon/intron fate of any given RNA sequence. At present, however, what we still lack is a precise understanding of how all these processes add up to help the spliceosome reach a decision. Therefore, it is expected that future challenges in splicing research will be the careful characterization of all these influences to improve our ability to predict splicing choices in different organisms or in specific contexts. WIREs RNA 2013, 4:49–60. doi: 10.1002/wrna.1140

Cellular Model of TAR DNA-binding Protein 43 (TDP-43) Aggregation Based on Its C-terminal Gln/Asn-rich Region

Background: TDP-43 is the principal protein component of cellular inclusion in ALS and FTLD. Results: Tandem repetitions of TDP-43 residues 331–369 induce cellular aggregates that recruit endogenous TDP-43. Conclusion: Our results establish a cell-based TDP-43 aggregation model. Significance: This model will be useful to investigate TDP-43 aggregation and develop strategies/effectors able to prevent/reduce this phenomenon. TDP-43 is one of the major components of the neuronal and glial inclusions observed in several neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. These characteristic aggregates are a “landmark” of the disease, but their role in the pathogenesis is still obscure. In previous works, we have shown that the C-terminal Gln/Asn-rich region (residues 321–366) of TDP-43 is involved in the interaction of this protein with other members of the heterogeneous nuclear ribonucleoprotein protein family. Furthermore, we have shown that the interaction through this region is important for TDP-43 splicing inhibition of cystic fibrosis transmembrane regulator exon 9, and there were indications that it was involved in the aggregation process. Our experiments show that in cell lines and primary rat neuronal cultures, the introduction of tandem repeats carrying the 331–369-residue Gln/Asn region from TDP-43 can trigger the formation of phosphorylated and ubiquitinated aggregates that recapitulate many but not all the characteristics observed in patients. These results establish a much needed cell-based TDP-43 aggregation model useful to investigate the mechanisms involved in the formation of inclusions and the gain- and loss-of-function consequences of TDP-43 aggregation within cells. In addition, it will be a powerful tool to test novel therapeutic strategies/effectors aimed at preventing/reducing this phenomenon.

NF1 mRNA biogenesis: Effect of the genomic milieu in splicing regulation of the NF1 exon 37 region

We have studied the splicing regulation of NF1 exons 36 and 37. We show that they not only require an intact exonic Splicing Enhancer (ESE) within exon 37, but also need the genomic region stretching from exons 31 to 38. Any nucleotide change in two exon 37 third codon positions disrupts the ESE. The extent of exons 36 and 37 skipping due to a mutated ESE depends on the genomic context. This is a unique example of what may be a more general phenomena involved in the tuning of pre‐mRNA processing and gene expression modulation in the chromosomal setting.

TDP-43 affects splicing profiles and isoform production of genes involved in the apoptotic and mitotic cellular pathways

In recent times, high-throughput screening analyses have broadly defined the RNA cellular targets of TDP-43, a nuclear factor involved in neurodegeneration. A common outcome of all these studies is that changing the expression levels of this protein can alter the expression of several hundred RNAs within cells. What still remains to be clarified is which changes represent direct cellular targets of TDP-43 or just secondary variations due to the general role played by this protein in RNA metabolism. Using an HTS-based splicing junction analysis we identified at least six bona fide splicing events that are consistent with being controlled by TDP-43. Validation of the data, both in neuronal and non-neuronal cell lines demonstrated that TDP-43 substantially alters the levels of isoform expression in four genes potentially important for neuropathology: MADD/IG20, STAG2, FNIP1 and BRD8. For MADD/IG20 and STAG2, these changes could also be confirmed at the protein level. These alterations were also observed in a cellular model that successfully mimics TDP-43 loss of function effects following its aggregation. Most importantly, our study demonstrates that cell cycle alterations induced by TDP-43 knockdown can be recovered by restoring the STAG2, an important component of the cohesin complex, normal splicing profile.

Predominance of spliceosomal complex formation over polyadenylation site selection in TDP-43 autoregulation

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.

Neurodegeneration and RNA‐binding proteins

In the eukaryotic nucleus, RNA‐binding proteins (RBPs) play a very important role in the life cycle of both coding and noncoding RNAs. As soon as they are transcribed, in fact, all RNA molecules within a cell are bound by distinct sets of RBPs that have the task of regulating its correct processing, transport, stability, and function/translation up to its final degradation. These tasks are particularly important in cells that have a complex RNA metabolism, such as neurons. Not surprisingly, therefore, recent findings have shown that the misregulation of genes involved in RNA metabolism or the autophagy/proteasome pathway plays an important role in the onset and progression of several neurodegenerative diseases. In this article, we aim to review the recent advances that link neurodegenerative processes and RBP proteins. WIREs RNA 2017, 8:e1394. doi: 10.1002/wrna.1394

Functional Analysis of Mutations in Exon 9 of NF1 Reveals the Presence of Several Elements Regulating Splicing.

Neurofibromatosis type 1 (NF1) is one of the most common human hereditary disorders, predisposing individuals to the development of benign and malignant tumors in the nervous system, as well as other clinical manifestations. NF1 is caused by heterozygous mutations in the NF1 gene and around 25% of the pathogenic changes affect pre-mRNA splicing. Since the molecular mechanisms affected by these mutations are poorly understood, we have analyzed the splicing mutations identified in exon 9 of NF1, which is particularly prone to such changes, to better define the possible splicing regulatory elements. Using a minigene approach, we studied the effect of five splicing mutations in this exon described in patients. These highlighted three regulatory motifs within the exon. An in vivo splicing analysis of an extensive collection of changes generated in the minigene demonstrated that the CG motif at c.910-911 is critical for the recognition of exon 9. We also found that the GC motif at c.945-946 is involved in exon recognition through SRSF2 and that this motif is part of a Composite Exon Splicing Regulatory Element made up of physically overlapping enhancer and silencer elements. Finally, through an in vivo splicing analysis and in vitro binding assays, we demonstrated that the c.1007G>A mutation creates an Exonic Splicing Silencer element that binds the hnRNPA1 protein. The complexity of the splicing regulatory elements present in exon 9 is most likely responsible for the fact that mutations in this region represent 25% of all exonic changes that affect splicing in the NF1 gene.

Characterization of Variegate Porphyria Mutations Using a Minigene Approach

Porphyrias are a group of metabolic diseases that affect the skin and/or nervous system. In 2008, three unrelated patients were diagnosed with variegate porphyria at the CIPYP (Centro de Investigaciones sobre Porfirinas y Porfirias). Sequencing of the protoporphyrinogen oxidase gene, the gene altered in this type of porphyria, revealed three previously undescribed mutations: c.338+3insT, c.807G>A, and c.808-1G>C. As these mutations do not affect the protein sequence, we hypothesized that they might be splicing mutations. RT-PCRs performed on the patients mRNAs showed normal mRNA or no amplification at all. This result indicated that the aberrant spliced transcript is possibly being degraded. In order to establish whether they were responsible or not for the patients disease by causing aberrant splicing, we utilized a minigene approach. We found that the three mutations lead to exon skipping; therefore, the abnormal mRNAs are most likely degraded by a mechanism such as nonsense-mediated decay. In conclusion, these mutations are responsible for the disease because they alter the normal splicing pathway, thus providing a functional explanation for the appearance of disease and highlighting the use of minigene assays to complement transcript analysis.