Npl3 functions in mRNP assembly by recruitment of mRNP components to the transcription site and their transfer onto the mRNA.

RNA-binding proteins (RBPs) control every RNA metabolic process by multiple protein-RNA and protein-protein interactions. Their roles have largely been analyzed by crude mutations, which abrogate multiple functions at once and likely impact the structural integrity of the large ribonucleoprotein particles (RNPs) these proteins function in. Using UV-induced RNA-protein crosslinking of entire cells, protein complex purification and mass spectrometric analysis, we identified >100 in vivo RNA crosslinks in 16 nuclear mRNP components in Saccharomyces cerevisiae. For functional analysis, we chose Npl3, which displayed crosslinks in its two RNA recognition motifs (RRMs) and in the connecting flexible linker region. Both RRM domains and the linker uniquely contribute to RNA recognition as revealed by NMR and structural analyses. Interestingly, mutations in these regions cause different phenotypes, indicating distinct functions of the different RNA-binding domains. Notably, an npl3-Linker mutation strongly impairs recruitment of several mRNP components to chromatin and incorporation of other mRNP components into nuclear mRNPs, establishing a so far unknown function of Npl3 in nuclear mRNP assembly. Taken together, our integrative analysis uncovers a specific function of the RNA-binding activity of the nuclear mRNP component Npl3. This approach can be readily applied to RBPs in any RNA metabolic process.

The COP9 Signalosome regulates seed germination by facilitating protein degradation of RGL2 and ABI5.

The control of seed germination and seed dormancy are critical for the successful propagation of plant species, and are important agricultural traits. Seed germination is tightly controlled by the balance of gibberellin (GA) and abscisic acid (ABA), and is influenced by environmental factors. The COP9 Signalosome (CSN) is a conserved multi-subunit protein complex that is best known as a regulator of the Cullin-RING family of ubiquitin E3 ligases (CRLs). Multiple viable mutants of the CSN showed poor germination, except for csn5b-1. Detailed analyses showed that csn1-10 has a stronger seed dormancy, while csn5a-1 mutants exhibit retarded seed germination in addition to hyperdormancy. Both csn5a-1 and csn1-10 plants show defects in the timely removal of the germination inhibitors: RGL2, a repressor of GA signaling, and ABI5, an effector of ABA responses. We provide genetic evidence to demonstrate that the germination phenotype of csn1-10 is caused by over-accumulation of RGL2, a substrate of the SCF (CRL1) ubiquitin E3 ligase, while the csn5a-1 phenotype is caused by over-accumulation of RGL2 as well as ABI5. The genetic data are consistent with the hypothesis that CSN5A regulates ABI5 by a mechanism that may not involve CSN1. Transcriptome analyses suggest that CSN1 has a more prominent role than CSN5A during seed maturation, but CSN5A plays a more important role than CSN1 during seed germination, further supporting the functional distinction of these two CSN genes. Our study delineates the molecular targets of the CSN complex in seed germination, and reveals that CSN5 has additional functions in regulating ABI5, thus the ABA signaling pathway.

Improved Magnetization Transfers among Quadrupolar Nuclei in Two-Dimensional Homonuclear Correlation NMR Experiments Applied to Inorganic Network Structures.

We demonstrate that supercycles of previously introduced two-fold symmetry dipolar recoupling schemes may be utilized successfully in homonuclear correlation nuclear magnetic resonance (NMR) spectroscopy for probing proximities among half-integer spin quadrupolar nuclei in network materials undergoing magic-angle-spinning (MAS). These (SR2 2 1 ) M , (SR2 4 1 ) M , and (SR2 8 1 )M recoupling sequences with M = 3 and M = 4 offer comparably efficient magnetization transfers in single-quantum-single-quantum (1Q-1Q) correlation NMR experiments under moderately fast MAS conditions, as demonstrated at 14.1 T and 24 kHz MAS in the contexts of 11 B NMR on a Na 2 O-CaO-B 2 O 3 -SiO 2 glass and 27 Al NMR on the open framework aluminophosphate AlPO-CJ19 [(NH 4 ) 2 Al 4 (PO 4 ) 4 HPO 4 · H 2 O]. Numerically simulated magnetization transfers in spin-3/2 pairs revealed a progressively enhanced tolerance to resonance offsets and rf-amplitude errors of the recoupling pulses along the series (SR2 2 1 ) M < (SR2 4 1 ) M < (SR2 8 1 )M for increasing differences in chemical shifts between the two nuclei. Nonetheless, for scenarios of a relatively minor chemical-shift dispersions ( ≲ 3 kHz), the (SR2 2 1 )M supercycles perform best both experimentally and in simulations.

A diagnostic algorithm to distinguish desmoplastic from spindle cell melanoma.

Spindle cell melanoma and desmoplastic melanoma differ clinically in prognosis and therapeutic implications; however, because of partially overlapping histopathological features, diagnostic distinction of spindle cell from desmoplastic melanoma is not always straightforward. A direct comparison of diagnostic and therapeutic biomarkers has not been performed. Meta-review of the literature discloses key clinicopathological differences between spindle cell and desmoplastic melanoma, including immunophenotypes. Using 50 biomarkers available in routine diagnostics, we examined 38 archival cases (n=16 spindle, 18 desmoplastic, 4 mixed spindle/desmoplastic melanoma). S100 remains as the most reliable routine marker to reach the diagnosis of melanoma in spindle cell and desmoplastic melanoma. We identified nine distinctly labeling markers with spindle cell melanoma showing positivity for laminin, p75, HMB45, c-kit, and MelanA, and desmoplastic melanoma preferentially labeling with collagen IV, trichrome, CD68, and MDM2. On the basis of comparisons of test performance measures, MelanA and trichrome were used to devise a 94% sensitive diagnostic algorithm for the distinction of desmoplastic from spindle cell melanoma. Gene amplification and expression status was assessed for a set of potentially drugable targets (HER2, EGFR, MET, MDM2, TP53, ALK, MYC, FLI-1, and KIT). Fluorescent in situ hybridizations did not reveal a significant number of gene aberrations/rearrangements; however, protein overexpression for at least one of these markers was identified in 35 of 38 cases (92%). In addition, we found BRAF mutations in 31% of spindle cell and 5% of desmoplastic melanoma, with an overall mutation frequency of 16% (n=6/38). We present the first comprehensive screening study of diagnostic and therapeutic biomarkers in spindle cell and desmoplastic melanoma. The devised algorithm allows diagnostic distinction of desmoplastic from spindle cell melanoma when routine histology is not decisive.

Six-Month Follow-Up after Vaccination with BNT162b2: SARS-CoV-2 Antigen-Specific Cellular and Humoral Immune Responses in Hemodialysis Patients and Kidney Transplant Recipients.

Hemodialysis patients (HDP) and kidney transplant recipients (KTR) have a high risk of infection with SARS-CoV-2 with poor clinical outcomes. Because of this, vaccination of these groups of patients against SARS-CoV-2 is particularly important. However, immune responses may be impaired in immunosuppressed and chronically ill patients. Here, our aim was to compare the efficacy of an mRNA-based vaccine in HDP, KTR, and healthy subjects. DESIGN: In this prospective observational cohort study, the humoral and cellular response of prevalent 192 HDP, 50 KTR, and 28 healthy controls (HC) was assessed 1, 2, and 6 months after the first immunization with the BNT162b2 mRNA vaccine. RESULTS: After 6 months, 97.5% of HDP, 37.9% of KTR, and 100% of HC had an antibody response. Median antibody levels were 1539.7 (±3355.8), 178.5 (±369.5), and 2657.8 (±2965.8) AU/mL in HDP, KTR, and HC, respectively (p ≤ 0.05). A SARS-CoV-2 antigen-specific cell response to vaccination was found in 68.8% of HDP, 64.5% of KTR, and 90% of HC. CONCLUSION: The humoral response rates to mRNA-based vaccination of HDPs are comparable to HCs, but antibody titers are lower. Furthermore, HDPs have weaker T-cell response to vaccination than HCs. KTRs have very low humoral and antigen-specific cellular response rates and antibody titers, which requires other vaccination strategies in addition to booster vaccination.

Assessment of new symmetry-based dipolar recoupling schemes for homonuclear magnetization exchange between quadrupolar nuclei in two-dimensional correlation MAS NMR.

We provide an extensive experimental and numerical evaluation of MQ-phase (S)M supercycles with M={3,4} of three groups of symmetry-based homonuclear dipolar recoupling rf-pulse sequences, [Formula: see text] , for establishing proximities among half-integer spin quadrupolar nuclei under moderately fast magic-angle-spinning (MAS) conditions in single-quantum-single-quantum (1Q-1Q) correlation NMR experiments. The relative merits of the (S)M schemes for variations in resonance offsets and rf-amplitude errors were assessed by numerically simulated magnetization transfers in spin-3/2 pairs with variable isotropic chemical shifts and quadrupolar coupling constants. Experimental demonstrations of 23Na (spin-3/2) NMR on Na2MoO4·2H2O and 27Al (spin-5/2) NMR on AlPO-CJ19 [(NH4)2Al4(PO4)4HPO4·H2O] are presented at 14.1 T and 24 kHz MAS. We recommend using the (SR221)3 or (SR221)4 supercycles for samples that exhibit small chemical-shift dispersions (<3 kHz), and any (SRNNN/2)3 scheme with N⩾10 for larger spreads of isotropic chemical shifts. However, because the (SRNNN/2)3 sequences recouple heteronuclear dipolar interactions, their application to proton-bearing samples requires high-power proton decoupling during the mixing period. Alternatively, the (SR241)3 and (SR241)4 schemes may be employed in the absence of proton decoupling, but with poorer compensation to resonance-offsets and rf-amplitude errors.