Selective packaging of HIV-1 RNA genome is guided by the stability of 5′ untranslated region polyA stem

To generate infectious virus, HIV-1 must package two copies of its full-length RNA into particles. HIV-1 transcription initiates from multiple, neighboring sites, generating RNA species that only differ by a few nucleotides at the 5′ end, including those with one (1G) or three (3G) 5′ guanosines. Strikingly, 1G RNA is preferentially packaged into virions over 3G RNA. We investigated how HIV-1 distinguishes between these nearly identical RNAs using in-gel chemical probing combined with recently developed computational tools for determining RNA conformational ensembles, as well as cell-based assays to quantify the efficiency of RNA packaging into viral particles. We found that 1G and 3G RNAs fold into distinct structural ensembles. The 1G RNA, but not the 3G RNA, primarily adopts conformations with an intact polyA stem, exposed dimerization initiation site, and multiple, unpaired guanosines known to mediate Gag binding. Furthermore, we identified mutants that exhibited altered genome selectivity and packaged 3G RNA efficiently. In these mutants, both 1G and 3G RNAs fold into similar conformational ensembles, such that they can no longer be distinguished. Our findings demonstrate that polyA stem stability guides RNA-packaging selectivity. These studies also uncover the mechanism by which HIV-1 selects its genome for packaging: 1G RNA is preferentially packaged because it exposes structural elements that promote RNA dimerization and Gag binding.

MapToCleave: high-throughput profiling of microRNA biogenesis in living cells

Previous large-scale studies have uncovered many features that determine the processing of microRNA (miRNA) precursors, however, they have been conducted in vitro. Here we introduce MapToCleave, a new method to simultaneously profile processing of thousands of distinct RNA structures in living cells. Our new in cell method captures essentially all the biogenesis features that have been discovered through near two decades of in vitro studies - providing support for both approaches. We find that miRNA precursors with a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from twenty animal species. We systematically compare the importance of known and novel sequence and structural features and test biogenesis of miRNA precursors from ten animal and plant species in human cells. Lastly, we provide evidence that the GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryo-EM studies. In summary, we apply a new screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.

Reliability and validity of the Unified Classification System for postoperative periprosthetic femoral fractures around cemented polished taper-slip stems

Aims This aim of this study was to assess the reliability and validity of the Unified Classification System (UCS) for postoperative periprosthetic femoral fractures (PFFs) around cemented polished taper-slip (PTS) stems. Methods Radiographs of 71 patients with a PFF admitted consecutively at two centres between 25 February 2012 and 19 May 2020 were collated by an independent investigator. Six observers (three hip consultants and three trainees) were familiarized with the UCS. Each PFF was classified on two separate occasions, with a mean time between assessments of 22.7 days (16 to 29). Interobserver reliability for more than two observers was assessed using percentage agreement and Fleiss’ kappa statistic. Intraobserver reliability between two observers was calculated with Cohen kappa statistic. Validity was tested on surgically managed UCS type B PFFs where stem stability was documented in operation notes (n = 50). Validity was assessed using percentage agreement and Cohen kappa statistic between radiological assessment and intraoperative findings. Kappa statistics were interpreted using Landis and Koch criteria. All six observers were blinded to operation notes and postoperative radiographs. Results Interobserver reliability percentage agreement was 58.5% and the overall kappa value was 0.442 (moderate agreement). Lowest kappa values were seen for type B fractures (0.095 to 0.360). The mean intraobserver reliability kappa value was 0.672 (0.447 to 0.867), indicating substantial agreement. Validity percentage agreement was 65.7% and the mean kappa value was 0.300 (0.160 to 0.4400) indicating only fair agreement. Conclusion This study demonstrates that the UCS is unsatisfactory for the classification of PFFs around PTS stems, and that it has considerably lower reliability and validity than previously described for other stem types. Radiological PTS stem loosening in the presence of PFF is poorly defined and formal intraoperative testing of stem stability is recommended. Cite this article: Bone Joint J 2021;103-B(8):1339–1344.

The influence of some morphological parameters of the stem on the resistance to lodging of oat varieties and promising lines of the North-East FASC breeding

Resistance to stem lodging depends on the year conditions. The agro-climatic factors of the north-east of the European territory of the Russian Federation are unstable in terms of precipitation and temperature. Most of all, oat lodges in wet years, which is associated with an increase in plant length. To study the main morphological characters, the linear parameters of the stem and the indices of resistance to lodging were measured. The distribution of varieties and lines by plant length allowed to analyze the stem morphological parameters for each group. The main contribution to the length of plants is made by the upper and second internodes. A smaller proportion is accounted for by the length of the lower internode. The dependence of the resistance to lodging was revealed with the panicle length. The thicknesses of the first and second internodes affected some elements of the productivity structure (panicle length, panicle mass, the mass of 1000 grains, etc.). The Galchenko index (JG) and the ratio of the stem length to the diameter of the second internode (Lc/d2) had higher values in varieties with higher resistance to lodging. Promising lines 325h12, 200h15, 196h15, 175h15, 175h15 and the Sapsan variety combined stem stability and yield.