Defining Distinct RNA-Protein Interactomes of SARS-CoV-2 Genomic and Subgenomic RNAs.

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. Individual interactomes indicated viral associations with cell response pathways, including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We tested the significance of three protein interactors in these pathways (APOBEC3F, PPP1CC, and MSI2) using siRNA knockdowns, with several knockdowns affecting viral gene expression, most consistently PPP1CC. This study describes a new technology for high-resolution studies of SARS-CoV-2 RNA regulation and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs.

Host RNA binding proteins recognize viral RNA and play key roles in virus replication and antiviral defense mechanisms. SARS-CoV-2 generates a series of tiered subgenomic RNAs (sgRNAs), each encoding distinct viral protein(s) that regulate different aspects of viral replication. Here, for the first time, we demonstrate the successful isolation of SARS-CoV-2 genomic RNA and three distinct sgRNAs (N, S, and ORF8) from a single population of infected cells and characterize their protein interactomes. Over 500 protein interactors (including 260 previously unknown) were identified as associated with one or more target RNA at either of two time points. These included protein interactors unique to a single RNA pool and others present in multiple pools, highlighting our ability to discriminate between distinct viral RNA interactomes despite high sequence similarity. The interactomes indicated viral associations with cell response pathways including regulation of cytoplasmic ribonucleoprotein granules and posttranscriptional gene silencing. We validated the significance of five protein interactors predicted to exhibit antiviral activity (APOBEC3F, TRIM71, PPP1CC, LIN28B, and MSI2) using siRNA knockdowns, with each knockdown yielding increases in viral production. This study describes new technology for studying SARS-CoV-2 and reveals a wealth of new viral RNA-associated host factors of potential functional significance to infection.

Elucidating the RNA-Protein Interactomes of Target RNAs in Tissue.

RNA-protein interactions are key to many aspects of cellular homeostasis and their identification is important to understanding cellular function. Multiple strategies have been developed for the RNA-centric characterization of RNA-protein complexes. However, these studies have all been done in immortalized cell lines that do not capture the complexity of heterogeneous tissue samples. Here, we develop hybridization purification of RNA-protein complexes followed by mass spectrometry (HyPR-MS) for use in tissue samples. We isolated both polyadenylated RNA and the specific long noncoding RNA MALAT1 and characterized their protein interactomes. These results demonstrate the feasibility of HyPR-MS in tissue for the multiplexed characterization of specific RNA-protein complexes.

Organic dyes in illuminated manuscripts: a unique cultural and historic record.

In this study, we successfully addressed the challenges posed by the identification of dyes in medieval illuminations. Brazilwood pigment lakes and orcein purple colours were unequivocally identified in illuminated manuscripts dated by art historians to be from the thirteenth to the fifteenth centuries and in the Fernão Vaz Dourado Atlas (sixteenth century). All three works were on a parchment support. This was possible by combining Raman microscopy and surface-enhanced Raman spectroscopy with microspectrofluorimetry. To the best of our knowledge, this is the first time that brazilein, the main chromophore in brazilwood lake pigments, has been unequivocally identified by surface-enhanced Raman spectroscopy in an illuminated work (the Dourado Atlas). Complementing this identification, through microspectrofluorimetry and micro-Fourier transform infrared spectroscopy, it was possible to propose a complete paint formulation by comparison with our database of references; the dark pink hues, in the three case studies, were produced by combining brazilwood pigment lakes and gypsum in a protein- and gum arabic-based tempera. Orcein purple, also known as orchil dye, has been previously identified in medieval manuscripts, dated from the sixth to the ninth centuries. Our findings in fourteenth-sixteenth century manuscripts confirm the hypothesis that this dye was lost during the High Middle Ages, to be later rediscovered.This article is part of the themed issue 'Raman spectroscopy in art and archaeology'.

Orchil and Tyrian Purple: Two Centuries of Bedfords from Leeds.

A two-century-old archive relating to a Leeds dye manufacturer emerged a number of years ago from a Devon attic. Most items in the archive date from the mid-nineteenth century to the first quarter of the twentieth, a period of dye history when natural dyes overlapped with and were gradually replaced by synthetics. The archive contains material relevant to three generations of the Bedford family's manufacturing and research successes, as well as its close connections to the family of William Henry Perkin. A major portion of the archive's contents is connected to the trade in orchil, a purple-producing dye from a lichen on which the early fortunes of the company were based. A small envelope signed by Charles Samuel Bedford states that it contains "Tyrian Purple." After a historical and chemical investigation, we found that this dyestuff was indeed from mollusca, confirming that this vat-dyed textile sample is the most modern historic sample of Tyrian Purple yet found.

J. M. Despréaux' lichens from the Canary Islands and West Africa: an account of a 19th century collection found in an English archive.

This is an historical and descriptive account of 28 herbarium specimens, 27 lichens and an alga, found in the archives of Charles Chalcraft, a descendant of the Bedford family, who were dye manufacturers in Leeds, England, in the 19th century. The lichens comprise 13 different morphotypes collected in the Canary Islands and West Africa by the French botanist J. M. Despréaux between 1833 and 1839. The collections include samples of "Roccella fuciformis", "R. phycopsis" and "R. tinctoria" (including the fertile morphotype "R. canariensis"), "Ramalina crispatula" and "R. cupularis", two distinct morphotypes of "Sticta", "S. canariensis" and "S. dufouri", "Physconia enteroxantha", "Pseudevernia furfuracea var. ceratea" and "Pseudocyphellaria argyracea". The herbarium also includes authentic material of "Parmotrema tinctorum" and a probable syntype of "Seirophora scorigena". Most of these species are known as a source of the purple dye orchil, which was used to dye silk and wool.