In vivo analysis of NHPX reveals a novel nucleolar localization pathway involving a transient accumulation in splicing speckles

The NHPX protein is a nucleolar factor that binds directly to a conserved RNA target sequence found in nucleolar box C/D snoRNAs and in U4 snRNA. Using enhanced yellow fluorescent protein (EYFP)– and enhanced cyan fluorescent protein–NHPX fusions, we show here that NHPX is specifically accumulated in both nucleoli and Cajal bodies (CBs) in vivo. The fusion proteins display identical localization patterns and RNA binding specificities to the endogenous NHPX. Analysis of a HeLa cell line stably expressing EYFP–NHPX showed that the nucleolar accumulation of NHPX was preceded by its transient accumulation in splicing speckles. Only newly expressed NHPX accumulated in speckles, and the nucleolar pool of NHPX did not interchange with the pool in speckles, consistent with a unidirectional pathway. The transient accumulation of NHPX in speckles prior to nucleoli was observed in multiple cell lines, including primary cells that lack CBs. Inhibitor studies indicated that progression of newly expressed NHPX from speckles to nucleoli was dependent on RNA polymerase II transcription, but not on RNA polymerase I activity. The data show a specific temporal pathway involving the sequential and directed accumulation of NHPX in distinct subnuclear compartments, and define a novel mechanism for nucleolar localization

Quantifying argonaute proteins in and out of GW/P-bodies: Implications in microRNA activities

MicroRNAs (miRNAs) are a class of ∼22nt non-coding RNAs that regulate the translational potential and stability of mRNAs. Though constituting only 1-4% of human genes, miRNAs are predicted to regulate more than 60% of all mRNAs. The action of miRNAs is mediated through their associations with Argonaute proteins and mRNA targets. Previous studies indicated that though the majority of Argonaute proteins is diffusely distributed in the cytoplasm, a small fraction is consistently observed to be concentrated in a cytoplasmic compartment called GW/P-bodies. In this chapter, we will provide a quantitative and dynamic view of the subcellular localization of miRNA function, followed by a discussion on the possible roles of PBs in miRNA silencing.National Institutes of Health (U.S.) (Grant R01-CA133404)National Cancer Institute (U.S.) (Grant P01-CA42063)National Cancer Institute (U.S.) (Grant P30-CA14051

The Conserved Macrodomain Is a Potential Therapeutic Target for Coronaviruses and Alphaviruses

Emerging and re-emerging viral diseases pose continuous public health threats, and effective control requires a combination of non-pharmacologic interventions, treatment with antivirals, and prevention with vaccines. The COVID-19 pandemic has demonstrated that the world was least prepared to provide effective treatments. This lack of preparedness has been due, in large part, to a lack of investment in developing a diverse portfolio of antiviral agents, particularly those ready to combat viruses of pandemic potential. Here, we focus on a drug target called macrodomain that is critical for the replication and pathogenesis of alphaviruses and coronaviruses. Some mutations in alphavirus and coronaviral macrodomains are not tolerated for virus replication. In addition, the coronavirus macrodomain suppresses host interferon responses. Therefore, macrodomain inhibitors have the potential to block virus replication and restore the host’s protective interferon response. Viral macrodomains offer an attractive antiviral target for developing direct acting antivirals because they are highly conserved and have a structurally well-defined (druggable) binding pocket. Given that this target is distinct from the existing RNA polymerase and protease targets, a macrodomain inhibitor may complement current approaches, pre-empt the threat of resistance and offer opportunities to develop combination therapies for combating COVID-19 and future viral threats

Experimental Evidence for Simple Relations between Unpolarized and Polarized Parton Distributions

The Pauli exclusion principle is advocated for constructing the proton and neutron deep inelastic structure functions in terms of Fermi-Dirac distributions that we parametrize with very few parameters. It allows a fair description of the recent NMC data on $F^p_2(x,Q^2)$ and $F^n_2(x,Q^2)$ at $Q^2=4 GeV^2$, as well as the CCFR neutrino data at $Q^2=3$ and $5 GeV^2$. We also make some reasonable and simple assumptions to relate unpolarized and polarized quark parton distributions and we obtain, with no additional free parameters, the spin dependent structure functions $xg^p_1(x,Q^2)$ and $xg^n_1(x,Q^2)$. Using the correct $Q^2$ evolution, we have checked that they are in excellent agreement with the very recent SMC proton data at $Q^2=10 GeV^2$ and the SLAC neutron data at $Q^2=2 GeV^2$.Comment: 17 pages,CPT-94/P.3032,latex,6 fig available on cpt.univ-mrs.fr directory pub/preprints/94/fundamental-interactions /94-P.303

"I try and smile, I try and be cheery, I try not to be pushy. I try to say ‘I’m here for help’ but I leave feeling… worried’’: A qualitative study of perceptions of interactions with health professionals by community-based older adults with chronic pain

Background: Over 50% of community-dwelling older adults experience chronic pain, which threatens their quality of life. Of importance to their pain management is older people’s interaction with health professionals that, if unsatisfactory, may impair the outcome. Aims: To add to the limited research specific to older people living with chronic pain in the community, we explored how they perceive their experiences of interacting with health professionals, seeking factors that might optimise these interactions. Methods: Purposive sampling was used to recruit men and women .65 years with self-reported musculoskeletal chronic pain. Qualitative individual interviews and one group interview were undertaken with 23 participants. Data were transcribed verbatim and underwent Framework Analysis. Results: Three themes were identified. Seeking help illustrates issues around why older people in the community may or may not seek help for chronic pain, and highlights the potential involvement of social comparison. Importance of diagnosis illustrates the desire for professional validation of their condition and an aversion to vague explanations based on the person’s age. Being listened to and being heard illustrates the importance of empathic communication and understanding expectations, with due respect for the person’s age. Conclusions: In common with people of all ages, an effective partnership between an older person in pain and health professionals is essential if pain is to be reported, appropriately assessed and managed, because of the subjective nature of pain and its treatment responses. For older people with pain, perception about their age, by both parties in the partnership, is an additional factor that can unnecessarily interfere with the effectiveness of this partnership. Health professionals should engage with older adults to clarify their expectations about pain and its management, which may be influenced by perceptions about age; and to encourage expression of their concerns, which may also be affected by perceptions about age

Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs

MicroRNAs (miRNAs) are 19–22-nucleotide noncoding RNAs that post-transcriptionally regulate mRNA targets. We have identified endogenous miRNA binding sites in mouse embryonic stem cells (mESCs), by performing photo-cross-linking immunoprecipitation using antibodies to Argonaute (Ago2) followed by deep sequencing of RNAs (CLIP-seq). We also performed CLIP-seq in Dicer[superscript −/−] mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA dependent. A significantly enriched motif, GCACUU, was identified only in wild-type mESCs in 3′ untranslated and coding regions. This motif matches the seed of a miRNA family that constitutes ~68% of the mESC miRNA population. Unexpectedly, a G-rich motif was enriched in sequences cross-linked to Ago2 in both the presence and absence of miRNAs. Expression analysis and reporter assays confirmed that the seed-related motif confers miRNA-directed regulation on host mRNAs and that the G-rich motif can modulate this regulation.Leukemia & Lymphoma Society of AmericaUnited States. Public Health Service (Grant R01-GM34277)United States. Public Health Service (Grant R01-CA133404)National Cancer Institute (U.S.) (Grant P01-CA42063)National Cancer Institute (U.S.) Cancer Center Support (Grant P30-CA14051