In vitro and in vivo investigations on the interaction of bacterial RNase P with tRNA 3’-CCA

The Ribonuclease P (RNase P) is a ribonucleoprotein enzyme, which catalyses the 5’-maturation of precursor tRNAs. Bacterial RNase P consists of one RNA subunit (P RNA; encoded by rnpB; ~400 nt), and a protein subunit (P protein, encoded by rnpA; ~120 aa). In vitro under elevated salt concentrations the RNA subunit is catalytically active. However, under physiological conditions the protein subunit is essential for activity. Type A and B RNase P RNAs are interchangeable in vivo despite substantial biophysical differences It could be demonstrated that structural type A and type B bacterial RNase P RNAs can fully replace each other in vivo despite the many reported differences in their biogenesis, biochemical/biophysical properties and enzyme function in vitro. Even a single copy of E. coli rnpBwt integrated into the amyE site of the B. subtilis chromosome was sufficient for cell viability. The findings suggest that many of the reported idiosyncrasies of type A and B enzymes either do not reflect the in vivo situation or are not critical for RNase P function in vivo, at least under standard growth conditions. The precursor tRNA 3’-CCA interaction with Escherichia coli RNase P RNA is essential for catalysis by RNase P in vivo The L15 region of Escherichia coli RNase P RNA forms two Watson-Crick base pairs with precursor tRNA 3’-CCA termini (G292-C75 and G293-C74). Here, the phenotyes associated with disruption of the G292-C75 or G293-C74 pair in vivo was analyzed. Mutant RNase P RNA alleles (rnpBC292 and rnpBC293) caused severe growth defects in the E. coli rnpB mutant strain DW2 and abolished growth in the newly constructed mutant strain BW in which chromosomal rnpB expression strictly depended on the presence of arabinose. An isosteric C293-G74 base pair, but not a C292-G75 pair, fully restored catalytic performance in vivo, as shown for processing of precursor 4.5S RNA. This demonstrates that the base identity of G292, but not G293, contributes to the catalytic process in vivo. Activity assays with mutant RNase P holoenzymes assembled in vivo or in vitro revealed that the C292/293 mutations cause a severe functional defect at low Mg2+ concentrations (2 mM), which can be infered to be on the level of catalytically important Mg2+ recruitment. At 4.5 mM Mg2+, activity of mutant relative to the wild-type holoenzyme, was decreased only about 2-fold, but 13-24-fold at 2 mM Mg2+. Moreover, the findings make it unlikely that the C292/293 phenotypes include significant contributions from defects in protein binding, substrate affinity or RNA degradation. However, native PAGE experiments revealed non-identical RNA folding equilibria for the wild-type versus mutant RNase P RNAs, in a buffer- and preincubation-dependent manner. Thus, it cannot be excluded that altered folding of the mutant RNAs may have also contributed to their in vivo defect. In vivo role of bacterial type B RNase P interaction with tRNA 3’-CCA It has been unclear if catalysis by bacterial type B RNase P involves a specific interaction with p(recursor)tRNA 3’-CCA termini. We show that point mutations at two guanosines in loop L15 result in growth inhibition, which correlates with an enzyme defect at low Mg2+. For Bacillus subtilis RNase P, an isosteric C259-G74 bp fully and a C258-G75 bp slightly rescued catalytic proficiency, demonstrating Watson-Crick base-pairing to tRNA 3’-CCA and emphasizing the importance of G258 identity. We infer the defect of the mutant enzymes to be primarily on the level of recruitment of catalytically relevant Mg2+, with a possible contribution from altered RNA folding. Cell viability of bacteria expressing mutant RNase P RNAs could be (partially) restored by RNase P protein overexpression, resulting in increased cellular RNase P levels. Finally, we demonstrate that B. subtilis RNase P is able to cleave CCA-less ptRNAs in vivo. We conclude that the in vivo phenotype upon disruption of the CCA interaction is either due to a global deceleration in ptRNA maturation kinetics or severe blockage of 5’-maturation for a subset of ptRNAs

Another Look at Skidder Ride Vibration

Whole-body vibration data from recent tests on several log skidders are presented. Weighted RMS (root mean square) and RMQ (root mean quad) values are compared to FERIC results of 1987. The PSD (power spectral density) of the vertical motion of the seat base is compared with the PSDs currently defined in ISO and SAE Standards for the evaluation of seat suspensions on earthmoving equipment and agricultural tractors. This paper suggests the adoption of a seat performance test such as those contained in ISO 7096/ SAE J1385 for earthmoving equipment, or in ISO 5007/SAE J1386 for agricultural tractors. Seat suspensions capable of meeting the requirements of such a test will attenuate the vibration at the frequencies most prevalent on skidders, and should significantly improve the ride on these machines

Whiplash and Post-Concussion Syndrome following MVA

Background and Purpose: Neck pain is one of the most prevalent musculoskeletal conditions in today’s world affecting individuals of all ages. More specifically, Whiplash Associated Disorders (WAD) is a major classification of neck pain that is often caused by motor vehicle accidents (MVA). The purpose of this case report is to reflect and examine the available evidence relevant to the examination, evaluation, and treatment procedures that were used for a patient following a MVA. Case Description: The patient was a 52 year old female with COPD that presented with whiplash, post-concussion syndrome, and various soft tissue injuries following a motor vehicle accident. Intervention: The individual was seen by physical therapy for six days in the inpatient hospital setting followed by an additional 10 days in the transitional care unit (TCU). She received several physical therapy services including ice packs, interferential current (IFC) electrical stimulation, range of motion (ROM) activities, stretching and strengthening exercises, gait training, therapeutic ultrasound (US), and manual therapy. Outcomes: Ambulation distance increased from 15 feet using a front wheeled walker (FWW) on the initial evaluation to over 800 feet using a single point cane at discharge. Pain decreased from 10/10 on an ordinal scale on the first visit to an average of 4-7/10 throughout her final week at the facility. Strength was increased to functional levels as well as normal range of motion attained throughout all extremities Discussion: Active exercise, electrical stimulation, ultrasound, and manual therapy may be effective methods for improving pain and decreasing function

Genetic risk factors for neurodevelopmental disorders: insights from hiPSC-cerebral organoids

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder (NDD) characterized by remarkable phenotypic variability, where affected children manifest a spectrum of central nervous system (CNS) abnormalities, including brain tumors, impairments in attention, behavior, learning disabilities, and an increased incidence of autism spectrum disorder (ASD). A significant barrier to the implementation of precision medicine strategies for children with NF1 is a lack of prognostic risk factors to guide clinical management. However, emerging population-based genotype-phenotype association studies have suggested that the germline NF1 gene mutation may represent one clinically actionable risk factor for NF1-associated neurodevelopmental abnormalities. As a critical step in interpreting the significance of these reported genotype-phenotype correlations, we sought to determine whether germline NF1 gene mutations differentially affected human brain development, while controlling for other important confounding factors (e.g., sex, genomic differences). For these studies, we generated an isogenic series of CRISPR/Cas9-engineered human induced pluripotent stem cells (hiPSCs) harboring seven different intragenic NF1 patient germline NF1 gene mutations. Using this experimental platform, we established 2D neural progenitor cell (NPC) and astrocyte cultures, as well as three-dimensional (3D) human cerebral organoids (hCOs). While all mutations similarly increased proliferation and RAS activity in 2D NPCs and astrocytes, we observed striking differences between NF1 mutations on 2D NPC dopamine levels and 3D NPC proliferation, apoptosis, and neuronal differentiation in developing hCOs. Importantly, identical abnormalities were observed using patient-derived hiPSC 2D and 3D cultures bearing the same NF1 gene mutations, thus establishing differential effects of the germline NF1 gene mutation on human brain development. Based on these findings, we next sought to determine the cellular and molecular etiologies that underlie the severe developmental delays and intellectual disability (IQ \u3c 70) seen in children with a specific type of NF1 mutation involving heterozygous deletion of the NF1 gene and several contiguous genes (NF1-total gene deletion [NF1-TGD]). Using hCOs from three NF1 patients harboring NF1-TGDs, we identified both NPC proliferation and neuronal maturation abnormalities in NF1-TGD hCOs. While increased NPC proliferation resulted from decreased NF1/RAS regulation, the neuronal differentiation, survival, and maturation defects were caused by reduced cytokine receptor-like factor 3 (CRLF3) expression and impaired RhoA signaling. Furthermore, we demonstrated a higher autistic trait burden in NF1 patients harboring a deleterious germline mutation in the CRLF3 gene (c.1166T\u3eC, p.Leu389Pro). Taken together, this body of work conceptually advances the field by (1) demonstrating differential effects of NF1 mutations at the cellular and tissue levels in humans, establishing that the germline NF1 gene mutation is one important factor that underlies clinical variability in this monogenic syndrome, and (2) revealing a new causative gene within the NF1-TGD locus responsible for hCO neuronal abnormalities and autism in children with NF1

Minor changes largely restore catalytic activity of archaeal RNase P RNA from Methanothermobacter thermoautotrophicus

The increased protein proportion of archaeal and eukaryal ribonuclease (RNase) P holoenzymes parallels a vast decrease in the catalytic activity of their RNA subunits (P RNAs) alone. We show that a few mutations toward the bacterial P RNA consensus substantially activate the catalytic (C-) domain of archaeal P RNA from Methanothermobacter, in the absence and presence of the bacterial RNase P protein. Large increases in ribozyme activity required the cooperative effect of at least two structural alterations. The P1 helix of P RNA from Methanothermobacter was found to be extended, which increases ribozyme activity (ca 200-fold) and stabilizes the tertiary structure. Activity increases of mutated archaeal C-domain variants were more pronounced in the context of chimeric P RNAs carrying the bacterial specificity (S-) domain of Escherichia coli instead of the archaeal S-domain. This could be explained by the loss of the archaeal S-domain's capacity to support tight and productive substrate binding in the absence of protein cofactors. Our results demonstrate that the catalytic capacity of archaeal P RNAs is close to that of their bacterial counterparts, but is masked by minor changes in the C-domain and, particularly, by poor function of the archaeal S-domain in the absence of archaeal protein cofactors

Generation of human induced pluripotent stem cell-derived cerebral organoids for cellular and molecular characterization

Human induced pluripotent stem cell (hiPSC)-derived cerebral organoids (COs) can serve as a

Patient-derived iPSC-cerebral organoid modeling of the 17q11.2 microdeletion syndrome establishes CRLF3 as a critical regulator of neurogenesis

Neurodevelopmental disorders are often caused by chromosomal microdeletions comprising numerous contiguous genes. A subset of neurofibromatosis type 1 (NF1) patients with severe developmental delays and intellectual disability harbors such a microdeletion event on chromosome 17q11.2, involving the NF1 gene and flanking regions (NF1 total gene deletion [NF1-TGD]). Using patient-derived human induced pluripotent stem cell (hiPSC)-forebrain cerebral organoids (hCOs), we identify both neural stem cell (NSC) proliferation and neuronal maturation abnormalities in NF1-TGD hCOs. While increased NSC proliferation results from decreased NF1/RAS regulation, the neuronal differentiation, survival, and maturation defects are caused by reduced cytokine receptor-like factor 3 (CRLF3) expression and impaired RhoA signaling. Furthermore, we demonstrate a higher autistic trait burden in NF1 patients harboring a deleterious germline mutation in the CRLF3 gene (c.1166T\u3eC, p.Leu389Pro). Collectively, these findings identify a causative gene within the NF1-TGD locus responsible for hCO neuronal abnormalities and autism in children with NF1

Human iPSC-derived neurons and cerebral organoids establish differential effects of germline NF1 gene mutations

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by a spectrum of distinct germline NF1 gene mutations, traditionally viewed as equivalent loss-of-function alleles. To specifically address the issue of mutational equivalency in a disease with considerable clinical heterogeneity, we engineered seven isogenic human induced pluripotent stem cell lines, each with a different NF1 patient NF1 mutation, to identify potential differential effects of NF1 mutations on human central nervous system cells and tissues. Although all mutations increased proliferation and RAS activity in 2D neural progenitor cells (NPCs) and astrocytes, we observed striking differences between NF1 mutations on 2D NPC dopamine levels, and 3D NPC proliferation, apoptosis, and neuronal differentiation in developing cerebral organoids. Together, these findings demonstrate differential effects of NF1 gene mutations at the cellular and tissue levels, suggesting that the germline NF1 gene mutation is one factor that underlies clinical variability

Investigation of catalysis by bacterial RNase P via LNA and other modifications at the scissile phosphodiester

We analyzed cleavage of precursor tRNAs with an LNA, 2′-OCH3, 2′-H or 2′-F modification at the canonical (c0) site by bacterial RNase P. We infer that the major function of the 2′-substituent at nt −1 during substrate ground state binding is to accept an H-bond. Cleavage of the LNA substrate at the c0 site by Escherichia coli RNase P RNA demonstrated that the transition state for cleavage can in principle be achieved with a locked C3′ -endo ribose and without the H-bond donor function of the 2′-substituent. LNA and 2′-OCH3 suppressed processing at the major aberrant m−1 site; instead, the m+1 (nt +1/+2) site was utilized. For the LNA variant, parallel pathways leading to cleavage at the c0 and m+1 sites had different pH profiles, with a higher Mg2+ requirement for c0 versus m+1 cleavage. The strong catalytic defect for LNA and 2′-OCH3 supports a model where the extra methylene (LNA) or methyl group (2′-OCH3) causes a steric interference with a nearby bound catalytic Mg2+ during its recoordination on the way to the transition state for cleavage. The presence of the protein cofactor suppressed the ground state binding defects, but not the catalytic defects