Summary The conserved transcriptional regulator Heat Shock Factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol, yet its regulation is poorly understood. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC and Hsf1 senses an RQC-mediated translation stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.

Localized protein synthesis is a fundamental mechanism for creating distinct subcellular environments. Here we developed a generalizable proximity-specific ribosome profiling strategy that enables global analysis of translation in defined subcellular locations. We applied this approach to the endoplasmic reticulum (ER) in yeast and mammals. We observed the large majority of secretory proteins to be cotranslationally translocated, including substrates capable of post-translational insertion in vitro. Distinct translocon complexes engaged nascent chains at different points during synthesis. Whereas most proteins engaged the ER immediately after or even before signal sequence (SS) emergence, a class of Sec66-dependent proteins entered with a looped SS conformation. Finally, we observed rapid ribosome exchange into the cytosol after translation termination. These data provide insights into how distinct translocation mechanisms act in concert to promote efficient cotranslational recruitment.

Nearly all mitochondrial proteins are nuclear-encoded and are targeted to their mitochondrial destination from the cytosol. Here, we used proximity-specific ribosome profiling to comprehensively measure translation at the mitochondrial surface in yeast. Most inner membrane proteins were co-translationally targeted to mitochondria, reminiscent of proteins entering the endoplasmic reticulum (ER). Comparison between mitochondrial and ER localization demonstrated that the vast majority of proteins were targeted to a specific organelle. A prominent exception was the fumarate reductase Osm1, known to reside in mitochondria. We identified a conserved ER isoform of Osm1, which contributes to the oxidative protein folding capacity of the organelle. This dual localization was enabled by alternative translation initiation sites encoding distinct targeting signals. These findings highlight the exquisite in vivo specificity of organellar targeting mechanisms.

In the lactating breast, ERBB4 localizes to the nuclei of secretory epithelium while regulating activities of the signal transducer and activator of transcription (STAT) 5A transcription factor essential for milk-gene expression. We have identified an intrinsic ERBB4 NLS (residues 676–684) within the ERBB4 intracellular domain (4ICD) that is essential for nuclear accumulation of 4ICD. To determine the functional significance of 4ICD nuclear translocation in a physiologically relevant system, we have demonstrated that cotransfection of ERBB4 and STAT5A in a human breast cancer cell line stimulates β-casein promoter activity. Significantly, nuclear localization of STAT5A and subsequent stimulation of the β-casein promoter requires nuclear translocation of 4ICD. Moreover, 4ICD and STAT5A colocalize within nuclei of heregulin β1 (HRG)-stimulated cells and both proteins bind to the endogenous β-casein promoter in T47D breast cancer cells. Together, our results establish a novel molecular mechanism of transmembrane receptor signal transduction involving nuclear cotranslocation of the receptor intracellular domain and associated transcription factor. Subsequent binding of the two proteins at transcription factor target promoters results in activation of gene expression.

The unfolded protein response (UPR) monitors the protein folding capacity of the endoplasmic reticulum (ER). In all organisms analyzed to date, the UPR drives transcriptional programs that allow cells to cope with ER stress. The non-conventional splicing of Hac1 (yeasts) and XBP1 (metazoans) mRNA, encoding orthologous UPR transcription activators, is conserved and dependent on Ire1, an ER membrane-resident kinase/endoribonuclease. We found that the fission yeast Schizosaccharomyces pombe lacks both a Hac1/XBP1 ortholog and a UPR-dependent-transcriptional-program. Instead, Ire1 initiates the selective decay of a subset of ER-localized-mRNAs that is required to survive ER stress. We identified Bip1 mRNA, encoding a major ER-chaperone, as the sole mRNA cleaved upon Ire1 activation that escapes decay. Instead, truncation of its 3′ UTR, including loss of its polyA tail, stabilized Bip1 mRNA, resulting in increased Bip1 translation. Thus, S. pombe uses a universally conserved stress-sensing machinery in novel ways to maintain homeostasis in the ER.DOI: http://dx.doi.org/10.7554/eLife.00048.001

Although crosstalk between cell-surface and nuclear receptor signaling pathways has been implicated in the development and progression of endocrine-regulated cancers, evidence of direct coupling of these signaling pathways has remained elusive. Here we show that estrogen promotes an association between extranuclear estrogen receptor A (ER) and the epidermal growth factor receptor (EGFR) family member ERBB4. Ectopically expressed as well as endogenous ERBB4 interacts with and potentiates ER transactivation, indicating that the ERBB4/ER interaction is functional. Estrogen induces nuclear translocation of the proteolytic processed ERBB4 intracellular domain (4ICD) and nuclear translocation of 4ICD requires functional ligand-bound ER. The nuclear ER/4ICD complex is selectively recruited to estrogen-inducible gene promoters such as progesterone receptor (PgR) and stromal cell-derived factor 1 (SDF-1) but not to trefoil factor 1 precursor (pS2). Consistent with 4ICD-selective promoter binding, suppression of ERBB4 expression by interfering RNA shows that 4ICD coactivates ER transcription at the PgR and SDF-1 but not the pS2 promoter. Significantly, ERBB4 itself is an estrogen-inducible gene and the ERBB4 promoter harbors a consensus estrogen response element (ERE) half-site with overlapping activator protein-1 elements that bind ER and 4ICD in response to estrogen. Using a cell proliferation assay and a small interfering RNA approach, we show that ERBB4 expression is required for the growth-promoting action of estrogen in the T47D breast cancer cell line. Our results indicate that ERBB4 is a unique coregulator of ER, directly coupling extranuclear and nuclear estrogen actions in breast cancer. We propose that the contribution of an autocrine ERBB4/ER signaling pathway to tumor growth and therapeutic response should be considered when managing patients with ER-positive breast cancer. (Cancer Res 2006; 66(16): 7991-8)

Targeting RNAs with small molecules, a new frontier in drug discovery and development.

No abstract