Dataset Information

BS3 crosslinking of GRC3 and Las1

ABSTRACT: Ribosome assembly is a complex process involving the coordination of multiple enzymes. A recently discovered endoribonuclease (RNase), Las1, and the poly-nucleotide kinase (PNK), Grc3, assemble into a complex. To attempt to understand the coordination of the two enzymes in this complex, we performed chemical crosslinking and mass spectrometry and also solved a series of cryo-electron microscopy structures of RNase PNK in multiple conformational states.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Chaetomium Thermophilum

SUBMITTER: Jason Williams

LAB HEAD: Jason G. Williams

PROVIDER: PXD014738 | Pride | 2019-09-11

REPOSITORIES: Pride

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Publications

Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex.

Pillon Monica C MC Hsu Allen L AL Krahn Juno M JM Williams Jason G JG Goslen Kevin H KH Sobhany Mack M Borgnia Mario J MJ Stanley Robin E RE

Nature structural & molecular biology 20190905 9

Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of cells. The endoribonuclease (RNase) Las1 and the polynucleotide kinase (PNK) Grc3 assemble into a multienzyme complex, herein designated RNase PNK, to orchestrate processing of precursor ribosomal RNA (rRNA). RNase PNK belongs to the functionally diverse HEPN nuclease superfamily, whose members rely on distinct cues for nucle ...[more]

PMID: 31488907

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Project description:The subcellular localization of specific mRNAs is an evolutionary conserved mechanism that underlies the establishment of cellular polarity and specialized cell functions. In neurons, mRNA trafficking and local protein translation in dendrites provides an important mechanism that mediates synaptic development and plasticity. The significance of mRNA targeting and protein synthesis in axons, however, is still unclear. Only a small number of transcripts have been identified in axons to date, and their contribution to axon growth and neuronal survival remains largely unknown. Here, we report the results of a novel screen that allowed the separate identification of mRNAs localized in cell bodies and in axons of developing neurons. Using compartmentalized cultures of sympathetic neurons and Sequential Analysis of Gene Expression (SAGE), the screen identified more than 200 axonal mRNAs, including ones that encode cytoskeletal proteins and proteins that function in neural development and signal transduction. Importantly, several classes of transcripts were selectively enriched in axons, indicating that an active process drives the targeting of specific mRNAs from the cell bodies to the axons. This study is the first comprehensive and unbiased analysis of mRNA localization in subcellular domains of any neuronal cell type. We used compartmentalized chambers to culture neonatal rat sympathetic neurons (Campenot, 1977). In these cultures, the cell bodies are separated from the distal axons by a 1 mm wide Teflon divider, which maintains the cell bodies and axon terminals in separate fluid compartments. Primary rat sympathetic neurons are especially suitable for compartmentalized culture because they can be grown as a highly homogeneous population without glial cells. Neurons were seeded in the central compartment with nerve growth factor (NGF), and after a few days, the NGF was lowered in this compartment and supplied only to the peripheral compartment to stimulate axon growth. The anti-mitotic agent cytosine arabinoside C (Ara-C) was added to both compartments to remove non-neuronal cells. mRNA was then isolated after 12 days in culture (DIV) from cell body or axon compartments. As the initial mRNA content in axons was not sufficient to perform the SAGE analysis, both axon and cell body mRNAs were subjected to two rounds of linear amplification to obtain antisense RNA (aRNA). The amplified aRNA was then reverse transcribed and second strand synthesis was performed to proceed with the SAGE assay using the LongSAGE kit (Invitrogen) according to the manufacturer’s protocol.

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