RIP-Seq analysis of non-PPR chloroplast editing factors reveals broad RNA interactions and enrichment of less efficiently translated RNAs by OZ1 and ORRM1 complexes.

C-to-U RNA editing in angiosperm chloroplasts requires a large suite of proteins bound together in the editosome. The editosome is comprised of PPR proteins, RIP/MORFs, OZ proteins, and ORRM proteins that physically interact in high molecular weight complexes. The specific functions of non-PPR editing factors in the editosome are unclear, however, specific subsets of editing sites are affected by absence of non-PPR editing factors. Unlike the PPR components of editosomes that have predictable nucleotide specificities, domains present in non-PPR editing factors make RNA associations difficult to predict. In this study, chloroplast extracts were isolated from juvenile maize seedlings. RNAs were immunoprecipitated using polyclonal antibodies targeting non-PPR editing factors RIP9, OZ1, and ORRM1. RNA libraries from duplicate experiments were compared. RIP9 was associated with most of the non-ribosomal RNA content of chloroplasts, consistent with a general binding function to PPR L-motifs and tethering of large ribonucleoprotein complexes. The breadth of RNA associations was greater than predicted and include mRNAs without predicted editing sites, tRNA sequences, and introns. OZ1 and ORRM1 were associated with a highly similar pool of RNAs that have a bias toward lower translational efficiency values in mature chloroplasts. Lower translational efficiency was also associated with the pool of edited RNAs compared to RNAs without editing sites. The unexpected breadth of interactions by non-PPR editing factors suggests the editosome is large, diverse, and associated with RNAs with lower relative translational efficiency in mature chloroplasts.

Prevalencia de las úlceras por presión al egreso hospitalario en chile: tendencia del indicador 2001 al 2019

Objetivo: Analizar la tendencia de la prevalencia de úlceras por presión en Chile y sus regiones de acuerdo con los egresos hospitalarios. Material y Método: Estudio ecológico de series temporales, que analizó la prevalencia de úlceras por presión entre 2001 y 2019. Se realizó un análisis descriptivo, bivariante y lineal de tendencias. Este último con método de autorregresión Prais Winsten, calculando el cambio porcentual anual (APC) y sus intervalos de confianza al 95% (IC-95%). Resultados: La muestra fue de 11.060 casos. El 55,2% (6.103) fueron hombres, la media de edad fue 60 años (± 27.5) y la estancia hospitalaria fue 21,80 (± 35,084) días, siendo significativamente mayor en quienes presentaban lesiones por presión (p< 0,001). Existe una tendencia creciente y significativa en la prevalencia de úlceras por presión en Chile y todas sus regiones, teniendo promedio de un 11,33% de crecimiento interanual (APC= 0,0019; IC:95%= 0,0016-0,0022). Conclusión: Los resultados no son alentadores a pesar del aumento de la notificación de medidas de prevención y estandarización en los cuidados.

Objective: To analyze the trend in the prevalence of pressure ulcers in Chile and its regions according to hospital discharges. Material and Method: Ecological time series study, which analyzed the prevalence of pressure ulcers between 2001 and 2019. A descriptive, bivariate and linear analysis of trends was carried out. The latter with the Prais Winsten auto-regression method, calculating the annual percentage change (APC) and its 95% confidence intervals (95% CI). Results: The sample was 11,060 cases. 55.2% (6,103) were men, the average age was 60 years (± 27.5) and the hospital stay was 21.80 (± 35,084) days, being significantly longer in those with pressure injuries (p< 0.001). There is a growing and significant trend in the prevalence of pressure ulcers in Chile and all its regions, with an average of 11.33% interannual growth (APC= 0.0019; 95% CI= 0.0016-0.0022). Conclusion: The results are not encouraging despite the increase in notification of prevention measures and standardization of care.

Objetivo: Analisar a tendência da prevalência de úlceras por pressão no Chile e suas regiões de acordo com as altas hospitalares. Material e Método: Estudo ecológico de série temporal, que analisou a prevalência de úlceras por pressão entre 2001 e 2019. Foi realizada análise descritiva, bivariada e linear de tendências. Este último com o método de autorregressão de Prais Winsten, calculando a variação percentual anual (APC) e seus intervalos de confiança de 95% (IC 95%). Resultados: A amostra foi de 11.060 casos. 55,2% (6.103) eram homens, a idade média foi de 60 anos (± 27,5) e o tempo de internação foi de 21,80 (± 35.084) dias, sendo significativamente maior naqueles com lesão por pressão (p< 0,001). Há uma tendência crescente e significativa na prevalência de úlceras por pressão no Chile e em todas as suas regiões, com uma média de crescimento interanual de 11,33% (APC= 0,0019; IC 95%= 0,0016-0,0022). Conclusão: Os resultados não são animadores apesar do aumento da notificação de medidas de prevenção e da padronização dos cuidados.

A plant pentatricopeptide repeat protein with a DYW-deaminase domain is sufficient for catalyzing C-to-U RNA editing in vitro.

Pentatricopeptide repeat (PPR) proteins with C-terminal DYW domains are present in organisms that undergo C-to-U editing of organelle RNA transcripts. PPR domains act as specificity factors through electrostatic interactions between a pair of polar residues and the nitrogenous bases of an RNA target. DYW-deaminase domains act as the editing enzyme. Two moss (Physcomitrella patens) PPR proteins containing DYW-deaminase domains, PPR65 and PPR56, can convert Cs to Us in cognate, exogenous RNA targets co-expressed in Escherichia coli We show here that purified, recombinant PPR65 exhibits robust editase activity on synthetic RNAs containing cognate, mitochondrial PpccmFC sequences in vitro, indicating that a PPR protein with a DYW domain is solely sufficient for catalyzing C-to-U RNA editing in vitro Monomeric fractions possessed the highest conversion efficiency, and oligomeric fractions had reduced activity. Inductively coupled plasma (ICP)-MS analysis indicated a stoichiometry of two zinc ions per highly active PPR65 monomer. Editing activity was sensitive to addition of zinc acetate or the zinc chelators 1,10-o-phenanthroline and EDTA. Addition of ATP or nonhydrolyzable nucleotide analogs stimulated PPR65-catalyzed RNA-editing activity on PpccmFC substrates, indicating potential allosteric regulation of PPR65 by ATP. Unlike for bacterial cytidine deaminase, addition of two putative transition-state analogs, zebularine and tetrahydrouridine, failed to disrupt RNA-editing activity. RNA oligonucleotides with a single incorporated zebularine also did not disrupt editing in vitro, suggesting that PPR65 cannot bind modified bases due to differences in the structure of the active site compared with other zinc-dependent nucleotide deaminases.

Stable native RIP9 complexes associate with C-to-U RNA editing activity, PPRs, RIPs, OZ1, ORRM1 and ISE2.

The mitochondrial and chloroplast mRNAs of the majority of land plants are modified through cytidine to uridine (C-to-U) RNA editing. Previously, forward and reverse genetic screens demonstrated a requirement for pentatricopeptide repeat (PPR) proteins for RNA editing. Moreover, chloroplast editing factors OZ1, RIP2, RIP9 and ORRM1 were identified in co-immunoprecipitation (co-IP) experiments, albeit the minimal complex sufficient for editing activity was never deduced. The current study focuses on isolated, intact complexes that are capable of editing distinct sites. Peak editing activity for four sites was discovered in size-exclusion chromatography (SEC) fractions ≥ 670 kDa, while fractions estimated to be approximately 413 kDa exhibited the greatest ability to convert a substrate containing the editing site rps14 C80. RNA content peaked in the ≥ 670 kDa fraction. Treatment of active chloroplast extracts with RNase A abolished the relationship of editing activity with high-MW fractions, suggesting a structural RNA component in native complexes. By immunoblotting, RIP9, OTP86, OZ1 and ORRM1 were shown to be present in active gel filtration fractions, though OZ1 and ORRM1 were mainly found in low-MW inactive fractions. Active editing factor complexes were affinity-purified using anti-RIP9 antibodies, and orthologs to putative Arabidopsis thaliana RNA editing factor PPR proteins, RIP2, RIP9, RIP1, OZ1, ORRM1 and ISE2 were identified via mass spectrometry. Western blots from co-IP studies revealed the mutual association of OTP86 and OZ1 with native RIP9 complexes. Thus, RIP9 complexes were discovered to be highly associated with C-to-U RNA editing activity and other editing factors indicative of their critical role in vascular plant editosomes.