The UFM1 E3 ligase recognizes and releases 60S ribosomes from ER translocons.

Stalled ribosomes at the endoplasmic reticulum (ER) are covalently modified with the ubiquitin-like protein UFM1 on the 60S ribosomal subunit protein RPL26 (also known as uL24)1,2. This modification, which is known as UFMylation, is orchestrated by the UFM1 ribosome E3 ligase (UREL) complex, comprising UFL1, UFBP1 and CDK5RAP3 (ref. 3). However, the catalytic mechanism of UREL and the functional consequences of UFMylation are unclear. Here we present cryo-electron microscopy structures of UREL bound to 60S ribosomes, revealing the basis of its substrate specificity. UREL wraps around the 60S subunit to form a C-shaped clamp architecture that blocks the tRNA-binding sites at one end, and the peptide exit tunnel at the other. A UFL1 loop inserts into and remodels the peptidyl transferase centre. These features of UREL suggest a crucial function for UFMylation in the release and recycling of stalled or terminated ribosomes from the ER membrane. In the absence of functional UREL, 60S-SEC61 translocon complexes accumulate at the ER membrane, demonstrating that UFMylation is necessary for releasing SEC61 from 60S subunits. Notably, this release is facilitated by a functional switch of UREL from a 'writer' to a 'reader' module that recognizes its product-UFMylated 60S ribosomes. Collectively, we identify a fundamental role for UREL in dissociating 60S subunits from the SEC61 translocon and the basis for UFMylation in regulating protein homeostasis at the ER.

Targeted Single-Cell RNA and DNA Sequencing With Fluorescence-Activated Droplet Merger.

Analyzing every cell in a diverse sample provides insight into population-level heterogeneity, but abundant cell types dominate the analysis and rarer populations are scarcely represented in the data. To focus on specific cell types, the current paradigm is to physically isolate subsets of interest prior to analysis; however, it remains difficult to isolate and then single-cell sequence such populations because of compounding losses. Here, we describe an alternative approach that selectively merges cells with reagents to achieve enzymatic reactions without having to physically isolate cells. We apply this technique to perform single-cell transcriptome and genome sequencing of specific cell subsets. Our method for analyzing heterogeneous populations obviates the need for pre- or post-enrichment and simplifies single-cell workflows, making it useful for other applications in single-cell biology, combinatorial chemical synthesis, and drug screening.

A robust micro-electrospray ionization technique for high-throughput liquid chromatography/mass spectrometry proteomics using a sanded metal needle as an emitter.

A 60 microm internal diameter (i.d.) stainless-steel needle was adapted to the orthogonal ESI probe ( microESI) of a commercial ion trap mass spectrometer, and used for capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) protein identification experiments. The modification allows for the use of nitrogen sheath gas which helps in the nebulization at LC flow rates exceeding 500 nL/min and eliminates problems caused by liquid junctions commonly used to initiate nanospray ionization (NSI). A comparison is made between the performance of a 75 microm i.d. column with a 15 microm pulled glass tip using a liquid junction, and that of a 150 microm i.d. column using the new microESI device. The combination of the 150 microm i.d. column and microESI gave sensitivity close to that of NSI (250 attomoles horse heart myoglobin digest on column), and proved to be more robust than the standard pulled glass tips of similar i.d. No evidence of metal needle catalyzed oxidation of methionine was observed during analysis of the tetrapeptide MRFA under a range of test conditions. Phosphorylated peptides in a beta-casein tryptic digest were also successfully identified using the microESI interface with a steel needle. In addition it was found that a mild sanding of the metal needle tip improved spray performance.