Specific Nuclear Localizing Sequence Directs Two Myosin Isoforms to the Cell Nucleus in Calmodulin-Sensitive Manner

BACKGROUND: Nuclear myosin I (NM1) was the first molecular motor identified in the cell nucleus. Together with nuclear actin, they participate in crucial nuclear events such as transcription, chromatin movements, and chromatin remodeling. NM1 is an isoform of myosin 1c (Myo1c) that was identified earlier and is known to act in the cytoplasm. NM1 differs from the "cytoplasmic" myosin 1c only by additional 16 amino acids at the N-terminus of the molecule. This amino acid stretch was therefore suggested to direct NM1 into the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the mechanism of nuclear import of NM1 in detail. Using over-expressed GFP chimeras encoding for truncated NM1 mutants, we identified a specific sequence that is necessary for its import to the nucleus. This novel nuclear localization sequence is placed within calmodulin-binding motif of NM1, thus it is present also in the Myo1c. We confirmed the presence of both isoforms in the nucleus by transfection of tagged NM1 and Myo1c constructs into cultured cells, and also by showing the presence of the endogenous Myo1c in purified nuclei of cells derived from knock-out mice lacking NM1. Using pull-down and co-immunoprecipitation assays we identified importin beta, importin 5 and importin 7 as nuclear transport receptors that bind NM1. Since the NLS sequence of NM1 lies within the region that also binds calmodulin we tested the influence of calmodulin on the localization of NM1. The presence of elevated levels of calmodulin interfered with nuclear localization of tagged NM1. CONCLUSIONS/SIGNIFICANCE: We have shown that the novel specific NLS brings to the cell nucleus not only the "nuclear" isoform of myosin I (NM1 protein) but also its "cytoplasmic" isoform (Myo1c protein). This opens a new field for exploring functions of this molecular motor in nuclear processes, and for exploring the signals between cytoplasm and the nucleus

NMR-Based Analysis of Nanobodies to SARS-CoV-2 Nsp9 Reveals a Possible Antiviral Strategy Against COVID-19

Following the entry into the host cell, SARS-CoV-2 replication is mediated by the replication transcription complex (RTC) assembled through a number of nonstructural proteins (Nsps). A monomeric form of Nsp9 is particularly important for RTC assembly and function. In the present study, 136 unique nanobodies targeting Nsp9 are generated. Several nanobodies belonging to different B-cell lineages are expressed, purified, and characterized. Results from immunoassays applied to purified Nsp9 and neat saliva from coronavirus disease (COVID-19) patients show that these nanobodies effectively and specifically recognize both recombinant and endogenous Nsp9. Nuclear magnetic resonance analyses supported by molecular dynamics reveal a composite Nsp9 oligomerization pattern and demonstrate that both nanobodies stabilize the tetrameric form of wild-type Nsp9 also identifying the epitopes on the tetrameric assembly. These results can have important implications in the potential use of these nanobodies to combat viral replication

Myosin VI regulates the spatial organisation of mammalian transcription initiation

During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI (MVI) in the nanoscale organisation of RNAPII. We reveal that MVI in the nucleus acts as the molecular anchor that holds RNAPII in high density clusters. Perturbation of MVI leads to the disruption of RNAPII localisation, chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of MVI in the spatial regulation of gene expression

The Relevant Market: An Acceptable Limit to Competition Analysis?

Imagine that an agreement affects two separate markets. In market A, the agreement causes small (but appreciable) consumer welfare losses. In market B, the agreement generates massive consumer welfare benefits, dwarfing the losses in market A. Should one aggregate these costs and benefits across markets (‘Aggregate Across Markets’) when assessing the agreement under Article 101, or should one demand that the benefits outweigh the costs in each relevant market?This was one of three themes discussed at a breakfast roundtable that the OFT organised in May 2010. The focus of the debate was a recent OFT paper: OFT, Article 101(3) - A Discussion of Narrow versus Broad Definition of Benefits (OFT Discussion Paper). 23 experts attended, some from other UK competition authorities (the Competition Commission and Ofcom), DG COMP, some UK government departments (Department for Environment Food and Rural Affairs and the Department for Business Innovation and Skills), academia, businesses and law firms. The OFT compiled a synopsis of the roundtable’s discussion (Synopsis).The OFT Discussion Paper asks whether we should Aggregate Across Markets. The relevant Commission notice says: “The assessment under Article 81(3) [now Article 101(3)] of benefits flowing from restrictive agreements is in principle made within the confines of each relevant market to which the agreement relates.” The OFT Discussion Paper agrees that this is how the law currently stands. It claims that this issue is increasingly important, as two-sided markets become more prevalent.This paper starts with some preliminary observations on the OFT Discussion Paper, Section 2; then it shows that the EU Courts Aggregate Across Markets, so has the Commission, Section 3. Given how much it undermines legal certainty, the Commission needs strong justifications for deviating from the EU Courts’ case law in its guidance. The paper then discusses the advantages of Aggregating Across Markets, Section 4; and the disadvantages, Section 5. Section 6 concludes that, in addition to following the EU Courts’ case law; the arguments in favour of Aggregating Across Markets outweigh those against. I recommend that EU competition authorities that are not already Aggregating Across Markets, should start soon. As the techniques involved are no different from those used when one does not Aggregate Across Markets, decision-makers will easily be able to modify their behavior