Translation factor and RNA binding protein mRNA interactomes support broader RNA regulons for posttranscriptional control.

The regulation of translation provides a rapid and direct mechanism to modulate the cellular proteome. In eukaryotes, an established model for the recruitment of ribosomes to mRNA depends upon a set of conserved translation initiation factors. Nevertheless, how cells orchestrate and define the selection of individual mRNAs for translation, as opposed to other potential cytosolic fates, is poorly understood. We have previously found significant variation in the interaction between individual mRNAs and an array of translation initiation factors. Indeed, mRNAs can be separated into different classes based upon these interactions to provide a framework for understanding different modes of translation initiation. Here, we extend this approach to include new mRNA interaction profiles for additional proteins involved in shaping the cytoplasmic fate of mRNAs. This work defines a set of seven mRNA clusters, based on their interaction profiles with 12 factors involved in translation and/or RNA binding. The mRNA clusters share both physical and functional characteristics to provide a rationale for the interaction profiles. Moreover, a comparison with mRNA interaction profiles from a host of RNA binding proteins suggests that there are defined patterns in the interactions of functionally related mRNAs. Therefore, this work defines global cytoplasmic mRNA binding modules that likely coordinate the synthesis of functionally related proteins.

A Novel Flocked Swab Protocol Proves to Be an Effective Method for Culturing Elevator-Containing Endoscopes.

BACKGROUND: The challenging disinfection process for the elevator mechanism on duodenoscopes and linear echoendoscopes has been identified as a source of clinically significant bacterial transmission. Despite increased awareness, there continues to be a lack of definitive guidelines for bacterial culturing protocols for elevator-containing endoscopes. AIMS: To compare two different prospective bacterial surveillance protocols for duodenoscopes and linear echoendoscopes with regard to accuracy, efficiency, and cost. METHODS: Consecutive duodenoscopes and linear echoendoscopes used at a single tertiary care center were reprocessed following hospital and manufacturer guidelines, dried using an automatic endoscope-drying machine, and hung overnight in an upright position. Following reprocessing, culture samples were sequentially obtained from each endoscope using two methods, first, the brush protocol followed immediately by the swab protocol. RESULTS: A total of 532 primary cultures were collected from 17 duodenoscopes and eight linear echoendoscopes. Of these, 266 cultures gathered using the brush protocol were negative, while 266 cultures gathered using the swab protocol resulted in three positive cultures (1.1%). Positive cultures showed Enterobacter cloacae and Klebsiella pneumoniae from one duodenoscope and two linear echoendoscopes. Yearly, the brush protocol amounts to approximately 520 nursing hours, and the swab protocol takes an estimated 42 nursing hours. Annually, the swab protocol could save over $26,500 and 478 nursing hours. CONCLUSIONS: The proposed swab protocol was superior to the brush protocol when evaluating the presence of residual bacteria on elevator-containing endoscopes following reprocessing and saves cost and nursing hours.

Integrated multi-omics reveals common properties underlying stress granule and P-body formation.

Non-membrane-bound compartments such as P-bodies (PBs) and stress granules (SGs) play important roles in the regulation of gene expression following environmental stresses. We have systematically and quantitatively determined the protein and mRNA composition of PBs and SGs formed before and after nutrient stress. We find that high molecular weight (HMW) complexes exist prior to glucose depletion that we propose may act as seeds for further condensation of proteins forming mature PBs and SGs. We identify an enrichment of proteins with low complexity and RNA binding domains, as well as long, structured mRNAs that are poorly translated following nutrient stress. Many proteins and mRNAs are shared between PBs and SGs including several multivalent RNA binding proteins that promote condensate interactions during liquid-liquid phase separation. We uncover numerous common protein and RNA components across PBs and SGs that support a complex interaction profile during the maturation of these biological condensates. These interaction networks represent a tuneable response to stress, highlighting previously unrecognized condensate heterogeneity. These studies therefore provide an integrated and quantitative understanding of the dynamic nature of key biological condensates.

mRNA localization to P-bodies in yeast is bi-phasic with many mRNAs captured in a late Bfr1p-dependent wave.

The relocalization of translationally repressed mRNAs to mRNA processing bodies Pbodies is a key consequence of cellular stress across many systems. Pbodies harbor mRNA degradation components and are implicated in mRNA decay, but the relative timing and control of mRNA relocalization to Pbodies is poorly understood. We used the MS2GFP system to follow the movement of specific endogenous mRNAs in live Saccharomyces cerevisiae cells after nutritional stress. It appears that the relocalization of mRNA to Pbodies after stress is biphasic some mRNAs are present early, whereas others are recruited much later concomitant with recruitment of translation initiation factors, such as eIF4E. We also find that Bfr1p is a latephaselocalizing Pbody protein that is important for the delayed entry of certain mRNAS to Pbodies. Therefore, for the mRNAs tested, relocalization to Pbodies varies both in terms of the kinetics and factor requirements. This work highlights a potential new regulatory juncture in gene expression that would facilitate the overall rationalization of protein content required for adaptation to stress.

PKA isoforms coordinate mRNA fate during nutrient starvation.

A variety of stress conditions induce mRNA and protein aggregation into mRNA silencing foci, but the signalling pathways mediating these responses are still elusive. Previously we demonstrated that PKA catalytic isoforms Tpk2 and Tpk3 localise with processing and stress bodies in Saccharomyces cerevisiae. Here, we show that Tpk2 and Tpk3 are associated with translation initiation factors Pab1 and Rps3 in exponentially growing cells. Glucose starvation promotes the loss of interaction between Tpk and initiation factors followed by their accumulation into processing bodies. Analysis of mutants of the individual PKA isoform genes has revealed that the TPK3 or TPK2 deletion affects the capacity of the cells to form granules and arrest translation properly in response to glucose starvation or stationary phase. Moreover, we demonstrate that PKA controls Rpg1 and eIF4G(1) protein abundance, possibly controlling cap-dependent translation. Taken together, our data suggest that the PKA pathway coordinates multiple stages in the fate of mRNAs in association with nutritional environment and growth status of the cell.

Core Fermentation (CoFe) granules focus coordinated glycolytic mRNA localization and translation to fuel glucose fermentation.

Glycolysis is a fundamental metabolic pathway for glucose catabolism across biology, and glycolytic enzymes are among the most abundant proteins in cells. Their expression at such levels provides a particular challenge. Here we demonstrate that the glycolytic mRNAs are localized to granules in yeast and human cells. Detailed live cell and smFISH studies in yeast show that the mRNAs are actively translated in granules, and this translation appears critical for the localization. Furthermore, this arrangement is likely to facilitate the higher level organization and control of the glycolytic pathway. Indeed, the degree of fermentation required by cells is intrinsically connected to the extent of mRNA localization to granules. On this basis, we term these granules, core fermentation (CoFe) granules; they appear to represent translation factories, allowing high-level coordinated enzyme synthesis for a critical metabolic pathway.

Inhibition of translation initiation following glucose depletion in yeast facilitates a rationalization of mRNA content.

Glucose is the preferred carbon source for most eukaryotes and so it is important that cells can sense and react rapidly to fluctuations in glucose levels. It is becoming increasingly clear that the regulation of gene expression at the post-transcriptional level is important in the adaptation to changes in glucose levels, possibly as this could engender more rapid alterations in the concentrations of key proteins, such as metabolic enzymes. Following the removal of glucose from yeast cells a rapid inhibition of translation is observed. As a consequence, mRNPs (messenger ribonucleoproteins) relocalize into cytoplasmic granules known as P-bodies (processing bodies) and EGP-bodies. mRNA decay components localize into P-bodies, and thus these assemblies are likely to represent sites where mRNAs are targeted for degradation. In contrast, EGP-bodies lack any decay components and contain the eukaryotic translation initiation factors eIF4E, eIF4G and Pab1p, as well as other RNA-binding proteins. Therefore EGP-bodies probably constitute sites where mRNAs are earmarked for storage. So, it is possible that cells distinguish between transcripts and target them to either P-bodies or EGP-bodies depending on their functional value. The localization of mRNAs into these granules following glucose starvation may serve to preserve mRNAs that are involved in the diauxic shift to ethanol growth and entry into stationary phase, as well as to degrade mRNAs that are solely involved in glucose fermentation.

Translation factor mRNA granules direct protein synthetic capacity to regions of polarized growth.

mRNA localization serves key functions in localized protein production, making it critical that the translation machinery itself is present at these locations. Here we show that translation factor mRNAs are localized to distinct granules within yeast cells. In contrast to many messenger RNP granules, such as processing bodies and stress granules, which contain translationally repressed mRNAs, these granules harbor translated mRNAs under active growth conditions. The granules require Pab1p for their integrity and are inherited by developing daughter cells in a She2p/She3p-dependent manner. These results point to a model where roughly half the mRNA for certain translation factors is specifically directed in granules or translation factories toward the tip of the developing daughter cell, where protein synthesis is most heavily required, which has particular implications for filamentous forms of growth. Such a feedforward mechanism would ensure adequate provision of the translation machinery where it is to be needed most over the coming growth cycle.

Anti-α4ß7 therapy targets lymphoid aggregates in the gastrointestinal tract of HIV-1-infected individuals.

Gut homing CD4+ T cells expressing the integrin α4ß7 are early viral targets and contribute to HIV-1 pathogenesis, likely by seeding the gastrointestinal (GI) tract with HIV. Although simianized anti-α4ß7 monoclonal antibodies have shown promise in preventing or attenuating the disease course of simian immunodeficiency virus in nonhuman primate studies, the mechanisms of drug action remain elusive. We present a cohort of individuals with mild inflammatory bowel disease and concomitant HIV-1 infection receiving anti-α4ß7 treatment. By sampling the immune inductive and effector sites of the GI tract, we have discovered that anti-α4ß7 therapy led to a significant and unexpected attenuation of lymphoid aggregates, most notably in the terminal ileum. Given that lymphoid aggregates serve as important sanctuary sites for maintaining viral reservoirs, their attrition by anti-α4ß7 therapy has important implications for HIV-1 therapeutics and eradication efforts and defines a rational basis for the use of anti-α4ß7 therapy in HIV-1 infection.

Granules harboring translationally active mRNAs provide a platform for P-body formation following stress.

The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems.

Glucose depletion inhibits translation initiation via eIF4A loss and subsequent 48S preinitiation complex accumulation, while the pentose phosphate pathway is coordinately up-regulated.

Cellular stress can globally inhibit translation initiation, and glucose removal from yeast causes one of the most dramatic effects in terms of rapidity and scale. Here we show that the same rapid inhibition occurs during yeast growth as glucose levels diminish. We characterize this novel regulation showing that it involves alterations within the 48S preinitiation complex. In particular, the interaction between eIF4A and eIF4G is destabilized, leading to a temporary stabilization of the eIF3-eIF4G interaction on the 48S complex. Under such conditions, specific mRNAs that are important for the adaptation to the new conditions must continue to be translated. We have determined which mRNAs remain translated early after glucose starvation. These experiments enable us to provide a physiological context for this translational regulation by ascribing defined functions that are translationally maintained or up-regulated. Overrepresented in this class of mRNA are those involved in carbohydrate metabolism, including several mRNAs from the pentose phosphate pathway. Our data support a hypothesis that a concerted preemptive activation of the pentose phosphate pathway, which targets both mRNA transcription and translation, is important for the transition from fermentative to respiratory growth in yeast.