Shu-Bing Qian

Monitoring cotranslational protein folding in mammalian cells at codon resolution

How the ribosome-bound nascent chain folds to assume its functional tertiary structure remains a central puzzle in biology. In contrast to refolding of a denatured protein, cotranslational folding is complicated by the vectorial nature of nascent chains, the frequent ribosome pausing, and the cellular crowdedness. Here, we present a strategy called folding-associated cotranslational sequencing that enables monitoring of the folding competency of nascent chains during elongation at codon resolution. By using an engineered multidomain fusion protein, we demonstrate an efficient cotranslational folding immediately after the emergence of the full domain sequence. We also apply folding-associated cotranslational sequencing to track cotranslational folding of hemagglutinin in influenza A virus-infected cells. In contrast to sequential formation of distinct epitopes, the receptor binding domain of hemagglutinin follows a global folding route by displaying two epitopes simultaneously when the full sequence is available. Our results provide direct evidence of domain-wise global folding that occurs cotranslationally in mammalian cells.

Quantitating protein synthesis, degradation, and endogenous antigen processing.

Using L929 cells, we quantitated the macroeconomics of protein synthesis and degradation and the microeconomics of producing MHC class I associated peptides from viral translation products. To maintain a content of 2.6 x 10(9) proteins, each cells 6 x 10(6) ribosomes produce 4 x 10(6) proteins min(-1). Each of the cells 8 x 10(5) proteasomes degrades 2.5 substrates min(-1), creating one MHC class I-peptide complex for each 500-3000 viral translation products degraded. The efficiency of complex formation is similar in dendritic cells and macrophages, which play a critical role in activating T cells in vivo. Proteasomes create antigenic peptides at different efficiencies from two distinct substrate pools: rapidly degraded newly synthesized proteins that clearly represent defective ribosomal products (DRiPs) and a less rapidly degraded pool in which DRiPs may also predominate.

Decoding Human Cytomegalovirus

Dissecting HCMV Gene Expression Most of us are infected with human cytomegalovirus (HCMV), but severe disease is almost always limited to immunocompromised individuals or newborn infants. The virus has a relatively large (∼240 kb) DNA genome and shows a complex pattern of gene transcription, hinting at a complex regulatory and coding capacity. Stern-Ginossar et al. (p. 1088) mapped ribosome positions on HCMV transcripts during the course of viral infection of human fibroblast cells. The data suggest the presence of novel open reading frames (ORFs) lying within existing ORFs; very short ORFs upstream of canonical ORFs; ORFs antisense to canonical ORFs; and short, conserved ORFs encoded by long RNAs. Select ORFs were translated, dramatically expanding the coding capacity of the HCMV genome. A closer look at the human cytomegalovirus genome uncovers many new open reading frames. The human cytomegalovirus (HCMV) genome was sequenced 20 years ago. However, like those of other complex viruses, our understanding of its protein coding potential is far from complete. We used ribosome profiling and transcript analysis to experimentally define the HCMV translation products and follow their temporal expression. We identified hundreds of previously unidentified open reading frames and confirmed a fraction by means of mass spectrometry. We found that regulated use of alternative transcript start sites plays a broad role in enabling tight temporal control of HCMV protein expression and allowing multiple distinct polypeptides to be generated from a single genomic locus. Our results reveal an unanticipated complexity to the HCMV coding capacity and illustrate the role of regulated changes in transcript start sites in generating this complexity.

Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution

Understanding translational control in gene expression relies on precise and comprehensive determination of translation initiation sites (TIS) across the entire transcriptome. The recently developed ribosome-profiling technique enables global translation analysis, providing a wealth of information about both the position and the density of ribosomes on mRNAs. Here we present an approach, global translation initiation sequencing, applying in parallel the ribosome E-site translation inhibitors lactimidomycin and cycloheximide to achieve simultaneous detection of both initiation and elongation events on a genome-wide scale. This approach provides a view of alternative translation initiation in mammalian cells with single-nucleotide resolution. Systemic analysis of TIS positions supports the ribosome linear-scanning mechanism in TIS selection. The alternative TIS positions and the associated ORFs identified by global translation initiation sequencing are conserved between human and mouse cells, implying physiological significance of alternative translation. Our study establishes a practical platform for uncovering the hidden coding potential of the transcriptome and offers a greater understanding of the complexity of translation initiation.

CHIP-mediated stress recovery by sequential ubiquitination of substrates and Hsp70

Exposure of cells to various stresses often leads to the induction of a group of proteins called heat shock proteins (HSPs, molecular chaperones). Hsp70 is one of the most highly inducible molecular chaperones, but its expression must be maintained at low levels under physiological conditions to permit constitutive cellular activities to proceed. Heat shock transcription factor 1 (HSF1) is the transcriptional regulator of HSP gene expression, but it remains poorly understood how newly synthesized HSPs return to basal levels when HSF1 activity is attenuated. CHIP (carboxy terminus of Hsp70-binding protein), a dual-function co-chaperone/ubiquitin ligase, targets a broad range of chaperone substrates for proteasomal degradation. Here we show that CHIP not only enhances Hsp70 induction during acute stress but also mediates its turnover during the stress recovery process. Central to this dual-phase regulation is its substrate dependence: CHIP preferentially ubiquitinates chaperone-bound substrates, whereas degradation of Hsp70 by CHIP-dependent targeting to the ubiquitin–proteasome system occurs when misfolded substrates have been depleted. The sequential catalysis of the CHIP-associated chaperone adaptor and its bound substrate provides an elegant mechanism for maintaining homeostasis by tuning chaperone levels appropriately to reflect the status of protein folding within the cytoplasm.

Dynamic m6A mRNA methylation directs translational control of heat shock response

The most abundant mRNA post-transcriptional modification is N6-methyladenosine (m6A), which has broad roles in RNA biology. In mammalian cells, the asymmetric distribution of m6A along mRNAs results in relatively less methylation in the 5′ untranslated region (5′UTR) compared to other regions. However, whether and how 5′UTR methylation is regulated is poorly understood. Despite the crucial role of the 5′UTR in translation initiation, very little is known about whether m6A modification influences mRNA translation. Here we show that in response to heat shock stress, certain adenosines within the 5′UTR of newly transcribed mRNAs are preferentially methylated. We find that the dynamic 5′UTR methylation is a result of stress-induced nuclear localization of YTHDF2, a well-characterized m6A ‘reader’. Upon heat shock stress, the nuclear YTHDF2 preserves 5′UTR methylation of stress-induced transcripts by limiting the m6A ‘eraser’ FTO from demethylation. Remarkably, the increased 5′UTR methylation in the form of m6A promotes cap-independent translation initiation, providing a mechanism for selective mRNA translation under heat shock stress. Using Hsp70 mRNA as an example, we demonstrate that a single m6A modification site in the 5′UTR enables translation initiation independent of the 5′ end N7-methylguanosine cap. The elucidation of the dynamic features of 5′UTR methylation and its critical role in cap-independent translation not only expands the breadth of physiological roles of m6A, but also uncovers a previously unappreciated translational control mechanism in heat shock response.

Regulation of the Cytoplasmic Quality Control Protein Degradation Pathway by BAG2

The cytoplasm is protected against the perils of protein misfolding by two mechanisms: molecular chaperones (which facilitate proper folding) and the ubiquitin-proteasome system, which regulates degradation of misfolded proteins. CHIP (carboxyl terminus of Hsp70-interacting protein) is an Hsp70-associated ubiquitin ligase that participates in this process by ubiquitylating misfolded proteins associated with cytoplasmic chaperones. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. Using a proteomics approach, we have identified BAG2, a previously uncharacterized BAG domain-containing protein, as a common component of CHIP holocomplexes in vivo. Binding assays indicate that BAG2 associates with CHIP as part of a ternary complex with Hsc70, and BAG2 colocalizes with CHIP under both quiescent conditions and after heat shock. In vitro and in vivo ubiquitylation assays indicate that BAG2 is an efficient and specific inhibitor of CHIP-dependent ubiquitin ligase activity. This activity is due, in part, to inhibition of interactions between CHIP and its cognate ubiquitin-conjugating enzyme, UbcH5a, which may in turn be facilitated by ATP-dependent remodeling of the BAG2-Hsc70-CHIP heterocomplex. The association of BAG2 with CHIP provides a cochaperone-dependent regulatory mechanism for preventing unregulated ubiquitylation of misfolded proteins by CHIP.

Characterization of rapidly degraded polypeptides in mammalian cells reveals a novel layer of nascent protein quality control.

Approximately 30% of polypeptides synthesized by mammalian cells are degraded with a half-life of <10 min by proteasomes. These rapidly degraded polypeptides (RDPs) constitute the bulk of proteasome substrates and are the principal source of viral and self-peptide ligands for major histocompatibility complex class I molecules. Here we provide evidence that ∼75% of RDPs are degraded by the standard ubiquitin 26 S proteasome system and that their degradation is regulated by modulating Hsc70 activity in cells. Surprisingly, the remaining ∼25% of RDPs are degraded without ubiquitylation by 20 S proteasomes independently of 19 S regulators and in a manner that is largely unaffected by modulating Hsc70 activity. This latter pathway is utilized for generating an antigenic peptide from viral-defective ribosomal products. The dichotomy in the behavior of RDPs points to a novel quality control level for nascent proteins that is independent of the well established Hsc70-ubiquitin 26 S proteasome pathway.

Quantitative profiling of initiating ribosomes in vivo

Cells have evolved exquisite mechanisms to fine-tune the rate of protein synthesis in response to stress. Systemic mapping of start-codon positions and precise measurement of the corresponding initiation rate would transform our understanding of translational control. Here we present quantitative translation initiation sequencing (QTI-seq), with which the initiating ribosomes can be profiled in real time at single-nucleotide resolution. Resultant initiation maps not only delineated variations of start-codon selection but also highlighted a dynamic range of initiation rates in response to nutrient starvation. The integrated data set provided unique insights into principles of alternative translation and mechanisms controlling different aspects of translation initiation. With RiboTag mice, QTI-seq permitted tissue-specific profiling of initiating ribosomes in vivo. Liver cell–specific ribosome profiling uncovered a robust translational reprogramming of the proteasome system in fasted mice. Our findings illuminated the prevalence and dynamic nature of translational regulation pivotal to physiological adaptation in vivo.

Tight Linkage between Translation and MHC Class I Peptide Ligand Generation Implies Specialized Antigen Processing for Defective Ribosomal Products

There is mounting evidence that MHC class I peptide ligands are predominantly generated from defective ribosomal products and other classes of polypeptides degraded rapidly (t1/2 < 10 min) following their synthesis. The most direct evidence supporting this conclusion is the rapid inhibition of peptide ligand generation following cycloheximide-mediated inhibition of protein synthesis. In this study, we show that this linkage is due to depleting the pool of rapidly degraded proteins, and not to interference with other protein synthesis-dependent processes. Our findings indicate that in the model systems used in this study, MHC class I peptides are preferentially generated from rapidly degraded polypeptides relative to slowly degraded proteins. This conclusion is supported by the properties of peptide presentation from slowly degraded (t1/2 = 4 h) defective ribosomal products generated artificially by incorporation of the amino acid analog canavanine into a model viral Ag. We propose that specialized machinery exists to link protein synthesis with class I peptide ligand generation to enable the rapid detection of viral gene expression.