Youhna M. Ayala

TDP-43 binds heterogeneous nuclear ribonucleoprotein A/B through its C-terminal tail : An important region for the inhibition of cystic fibrosis transmembrane conductance regulator exon 9 splicing

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.

Structural determinants of the cellular localization and shuttling of TDP-43

TDP-43 (also known as TARDBP) regulates different processes of gene expression, including transcription and splicing, through RNA and DNA binding. Moreover, recent reports have shown that the protein interacts with the 3′UTRs of specific mRNAs. The aberrant cellular distribution and aggregation of TDP-43 were recently reported in neurodegenerative diseases, namely frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). A detailed description of the determinants for cellular localization has yet to emerge, including information on how the known functions of TDP-43 and cellular targeting affect each other. We provide the first experimental evidence that TDP-43 continuously shuttles between nucleus and cytoplasm in a transcription-dependent manner. Furthermore, we investigate the role of the functional TDP-43 domains in determining cellular targeting through a combination of immunofluorescence and biochemical fractionation methods. Our analyses indicate that the C-terminus is essential for solubility and cellular localization, because its deletion results in the formation of large nuclear and cytoplasmic aggregates. Disruption of the RNA-recognition domain required for RNA and DNA binding, however, alters nuclear distribution by decreasing TDP-43 presence in the nucleoplasm. Our findings suggest that TDP-43 solubility and localization are particularly sensitive to disruptions that extend beyond the newly found nuclear localization signal and depend on a combination of factors that are closely connected to the functional properties of this protein.

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.

The Na+ Binding Site of Thrombin

Thrombin is an allosteric serine protease existing in two forms, slow and fast, targeted toward anticoagulant and procoagulant activities. The slow → fast transition is induced by Na+ binding to a site contained within a cylindrical cavity formed by three antiparallel β-strands of the B-chain (Met-Tyr, Lys-Tyr, and Val-Gly) diagonally crossed by the Glu-Glu strand. The site is shaped further by the loop connecting the last two β-strands and is located more than 15 Å away from the catalytic triad. The cavity traverses through thrombin from the active site to the opposite surface and contains Asp of the primary specificity site near its midpoint. The bound Na+ is coordinated octahedrally by the carbonyl oxygen atoms of Tyr, Arg, and Lys, and by three highly conserved water molecules in the D-Phe-Pro-Arg chloromethylketone thrombin. The sequence in the Na+ binding loop is highly conserved in thrombin from 11 different species and is homologous to that found in other serine proteases involved in blood coagulation. Mutation of two Asp residues flanking Arg (D221A/D222K) almost abolishes the allosteric properties of thrombin and shows that the Na+ binding loop is also involved in direct recognition of protein C and antithrombin.

Depletion of TDP 43 overrides the need for exonic and intronic splicing enhancers in the human apoA-II gene

Exon 3 of the human apolipoprotein A-II (apoA-II) gene is efficiently included in the mRNA although its acceptor site is significantly weak because of a peculiar (GU)16 tract instead of a canonical polypyrimidine tract within the intron 2/exon 3 junction. Our previous studies demonstrated that the SR proteins ASF/SF2 and SC35 bind specifically an exonic splicing enhancer (ESE) within exon 3 and promote exon 3 splicing. In the present study, we show that the ESE is necessary only in the proper context. In addition, we have characterized two novel sequences in the flanking introns that modulate apoA-II exon 3 splicing. There is a G-rich element in intron 2 that interacts with hnRNPH1 and inhibits exon 3 splicing. The second is a purine rich region in intron 3 that binds SRp40 and SRp55 and promotes exon 3 inclusion in mRNA. We have also found that the (GU) repeats in the apoA-II context bind the splicing factor TDP-43 and interfere with exon 3 definition. Significantly, blocking of TDP-43 expression by small interfering RNA overrides the need for all the other cis-acting elements making exon 3 inclusion constitutive even in the presence of disrupted exonic and intronic enhancers. Altogether, our results suggest that exonic and intronic enhancers have evolved to balance the negative effects of the two silencers located in intron 2 and hence rescue the constitutive exon 3 inclusion in apoA-II mRNA.

TDP-43 regulates retinoblastoma protein phosphorylation through the repression of cyclin-dependent kinase 6 expression

TDP-43 (for TAR DNA binding protein) is a highly conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in specific pre-mRNA splicing and transcription events. TDP-43 recently has been identified as the main component of cytoplasmic inclusions in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), two neurodegenerative disorders. The cellular role of this protein remains to be identified. Here, we show that loss of TDP-43 results in dysmorphic nuclear shape, misregulation of the cell cycle, and apoptosis. Removal of TDP-43 in human cells significantly increases cyclin-dependent kinase 6 (Cdk6) protein and transcript levels. The control of Cdk6 expression mediated by TDP-43 involves GT repeats in the target gene sequence. Cdk6 up-regulation in TDP-43-depleted cells is accompanied by an increase in phosphorylation of two of its major targets, the retinoblastoma protein pRb and pRb-related protein pRb2/p130. TDP-43 silencing also is followed by changes in the expression levels of several factors that control cell proliferation. Morphological nuclear defects and increased apoptosis upon TDP-43 loss are mediated via the pRb pathway because pRb-negative cells (Saos-2) do not undergo programmed cell death or nuclear shape deformation upon TDP-43 removal. Our results identify a regulatory target of TDP-43 and show that TDP-43 depletion has important consequences in essential metabolic processes in human cells.

Functional mapping of the interaction between TDP-43 and hnRNP A2 in vivo

Nuclear factor TDP-43 has been reported to play multiple roles in transcription, pre-mRNA splicing, mRNA stability and mRNA transport. From a structural point of view, TDP-43 is a member of the hnRNP protein family whose structure includes two RRM domains flanked by the N-terminus and C-terminal regions. Like many members of this family, the C-terminal region can interact with cellular factors and thus serve to modulate its function. Previously, we have described that TDP-43 binds to several members of the hnRNP A/B family through this region. In this work, we set up a coupled minigene/siRNA cellular system that allows us to obtain in vivo data to address the functional significance of TDP-43-recruited hnRNP complex formation. Using this method, we have finely mapped the interaction between TDP-43 and the hnRNP A2 protein to the region comprised between amino acid residues 321 and 366. Our results provide novel details of protein–protein interactions in splicing regulation. In addition, we provide further insight on TDP-43 functional properties, particularly the lack of effects, as seen with our assays, of the disease-associated mutations that fall within the TDP-43 321-366 region: Q331K, M337V and G348C.

Molecular mechanisms of thrombin function

Abstract. The discovery of thrombin as a Na+-dependent allosteric enzyme has revealed a novel strategy for regulating protease activity and specificity. The allosteric nature of this enzyme influences all its physiologically important interactions and rationalizes a large body of structural and functional information. For the first time, a coherent mechanistic framework is available for understanding how thrombin interacts with fibrinogen, thrombomodulin and protein C, and how Na+ binding influences the specificity sites of the enzyme. This information can be used for engineering thrombin mutants with selective specificity towards protein C and for the rational design of potent active site inhibitors. Thrombin also serves as a paradigm for allosteric proteases. Elucidation of the molecular basis of the Na+-dependent allosteric regulation of catalytic activity, based on the residue present at position 225, provides unprecedented insights into the function and evolution of serine proteases. This mechanism represents one of the simplest and most important structure-function correlations ever reported for enzymes in general. All vitamin K-dependent proteases and some complement factors are subject to the Na+-dependent regulation discovered for thrombin. Na+ is therefore a key factor in the activation of zymogens in the coagulation and complement systems.

Characterization of the bacterial sensor protein PhoQ. Evidence for distinct binding sites for Mg2+ and Ca2+.

The PhoP/PhoQ two-component regulatory system governs several virulence properties in the Gram-negative bacterium Salmonella typhimurium. The PhoQ protein is a Mg2+ and Ca2+ sensor that modulates transcription of PhoP-regulated genes in response to the extracellular concentrations of these divalent cations. We have purified a 146-amino acid polypeptide corresponding to the periplasmic (i.e. sensing) domain of the PhoQ protein. Mg2+ altered the tryptophan intrinsic fluorescence of this polypeptide whereas Ba2+, which is unable to modulate transcription of PhoP-regulated genes, did not. Mg2+ was more effective than Ca2+ at repressing transcription of PhoP-activated genes in vivo. However, maximal repression was achieved when both cations were present. An avirulent mutant harboring a single amino acid substitution in the sensing domain of PhoQ exhibited lower affinity for Ca2+ but similar affinity for Mg2+. Cumulatively, these experiments demonstrate that Mg2+ can bind to the sensing domain of PhoQ and establish the presence of distinct binding sites for Mg2+ and Ca2+ in the PhoQ protein.

Molecular mapping of thrombin-receptor interactions

In addition to its procoagulant and anticoagulant roles in the blood coagulation cascade, thrombin works as a signaling molecule when it interacts with the G‐protein coupled receptors PAR1, PAR3, and PAR4. We have mapped the thrombin epitopes responsible for these interactions using enzymatic assays and Ala scanning mutagenesis. The epitopes overlap considerably, and are almost identical to those of fibrinogen and fibrin, but a few unanticipated differences are uncovered that help explain the higher (90‐fold) specificity of PAR1 relative to PAR3 and PAR4. The most critical residues for the interaction with the PARs are located around the active site where mutations affect recognition in the order PAR4 > PAR3 > PAR1. Other important residues for PAR binding cluster in a small area of exosite I where mutations affect recognition in the order PAR1 > PAR3 > PAR4. Owing to this hierarchy of effects, the mutation W215A selectively compromises PAR4 cleavage, whereas the mutation R67A abrogates the higher specificity of PAR1 relative to PAR3 and PAR4. 3D models of thrombin complexed with PAR1, PAR3, and PAR4 are constructed and account for the perturbations documented by the mutagenesis studies. Proteins 2001;45:107–116.