Compartmentalisation and localisation of the translation initiation factor (eIF) 4F complex in normally growing fibroblasts

Previous observations of association of mRNAs and ribosomes with subcellular structures highlight the importance of localised translation. However, little is known regarding associations between eukaryotic translation initiation factors and cellular structures within the cytoplasm of normally growing cells. We have used detergent-based cellular fractionation coupled with immunofluorescence microscopy to investigate the subcellular localisation in NIH3T3 fibroblasts of the initiation factors involved in recruitment of mRNA for translation, focussing on eIF4E, the mRNA cap-binding protein, the scaffold protein eIF4GI and poly(A) binding protein (PABP). We find that these proteins exist mainly in a soluble cytosolic pool, with only a subfraction tightly associated with cellular structures. However, this "associated" fraction was enriched in active "eIF4F" complexes (eIF4E.eIF4G.eIF4A.PABP). Immunofluorescence analysis reveals both a diffuse and a perinuclear distribution of eIF4G, with the perinuclear staining pattern similar to that of the endoplasmic reticulum. eIF4E also shows both a diffuse staining pattern and a tighter perinuclear stain, partly coincident with vimentin intermediate filaments. All three proteins localise to the lamellipodia of migrating cells in close proximity to ribosomes, microtubules, microfilaments and focal adhesions, with eIF4G and eIF4E at the periphery showing a similar staining pattern to the focal adhesion protein vinculin

Programmable in situ amplification for multiplexed imaging of mRNA expression

In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. With current approaches, it is challenging to simultaneously map multiple target mRNAs within whole-mount vertebrate embryos, representing a significant limitation in attempting to study interacting regulatory elements in systems most relevant to human development and disease. Here, we report a multiplexed fluorescent in situ hybridization method based on orthogonal amplification with hybridization chain reactions (HCR). With this approach, RNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled RNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample. Robust performance is achieved when imaging five target mRNAs simultaneously in fixed whole-mount and sectioned zebrafish embryos. HCR amplifiers exhibit deep sample penetration, high signal-to-background ratios and sharp signal localization

System wide analyses have underestimated protein abundances and the importance of transcription in mammals

Large scale surveys in mammalian tissue culture cells suggest that the protein expressed at the median abundance is present at 8,000–16,000 molecules per cell and that differences in mRNA expression between genes explain only 10–40% of the differences in protein levels. We find, however, that these surveys have significantly underestimated protein abundances and the relative importance of transcription. Using individual measurements for 61 housekeeping proteins to rescale whole proteome data from Schwanhausser et al. (2011), we find that the median protein detected is expressed at 170,000 molecules per cell and that our corrected protein abundance estimates show a higher correlation with mRNA abundances than do the uncorrected protein data. In addition, we estimated the impact of further errors in mRNA and protein abundances using direct experimental measurements of these errors. The resulting analysis suggests that mRNA levels explain at least 56% of the differences in protein abundance for the 4,212 genes detected by Schwanhausser et al. (2011), though because one major source of error could not be estimated the true percent contribution should be higher. We also employed a second, independent strategy to determine the contribution of mRNA levels to protein expression. We show that the variance in translation rates directly measured by ribosome profiling is only 9% of that inferred by Schwanhausser et al. (2011), and that the measured and inferred translation rates correlate poorly (R2 = 0.14). Based on this, our second strategy suggests that mRNA levels explain ∼84% of the variance in protein levels. We also determined the percent contributions of transcription, RNA degradation, translation and protein degradation to the variance in protein abundances using both of our strategies. While the magnitudes of the two estimates vary, they both suggest that transcription plays a more important role than the earlier studies implied and translation a much smaller role. Finally, the above estimates apply to those genes whose mRNA and protein expression was detected. Based on a detailed analysis by Hebenstreit et al. (2012), we estimate that approximately 40% of genes in a given cell within a population express no mRNA. Since there can be no translation in the absence of mRNA, we argue that differences in translation rates can play no role in determining the expression levels for the ∼40% of genes that are non-expressed

miR-1289 and “Zipcode”-like Sequence Enrich mRNAs in Microvesicles

Despite intensive studies, the molecular mechanisms by which the genetic materials are uploaded into microvesicles (MVs) are still unknown. This is the first study describing a zipcode-like 25 nucleotide (nt) sequence in the 3′-untranslated region (3′UTR) of mRNAs, with variants of this sequence present in many mRNAs enriched in MVs, as compared to their glioblastoma cells of origin. When this sequence was incorporated into the 3′UTR of a reporter message and expressed in a different cell type, it led to enrichment of the reporter mRNA in MVs. Critical features of this sequence are both a CUGCC core presented on a stem-loop structure and a miRNA-binding site, with increased levels of the corresponding miRNA in cells further increasing levels of mRNAs in MVs

The Actin Gene Family: Function Follows Isoform

Although actin is often thought of as a single protein, in mammals it actually consists of six different isoforms encoded by separate genes. Each isoform is remarkably similar to every other isoform, with only slight variations in amino acid sequence. Nevertheless, recent work indicates that actin isoforms carry out unique cellular functions. Here, we review evidence drawn from localization studies, mouse models, and biochemical characterization to suggest a model for how in vivo mixing of actin isoforms may influence cytoskeletal function in cells. © 2010 Wiley-Liss, Inc