Biology subjects

Cate, J.

Publications and source records attributed to Cate, J..

7 recordsLinked to original sources

bioRxiv2018

Kluyveromyces marxianus as a robust synthetic biology platform host

Throughout history, the yeast Saccharomyces cerevisiae has played a central role in human society due to its use in food production and more recently as a major industrial and model microorganism, because of the many genetic and genomic tools available to probe its biology. However S. cerevisiae has proven difficult to engineer to expand the carbon sources it can utilize, the products it can make, and the harsh conditions it can tolerate in industrial applications. Other yeasts that could solve many of these problems remain difficult to manipulate genetically. Here, we engineer the thermotolerant yeast Kluyveromyces marxianus to create a new synthetic biology platform. Using CRISPR-Cas9 mediated genome editing, we show that wild isolates of K. marxianus can be made heterothallic for sexual crossing. By breeding two of these mating-type engineered K. marxianus strains, we combined three complex traits- thermotolerance, lipid production, and facile transformation with exogenous DNA-into a single host. The ability to cross K. marxianus strains with relative ease, together with CRISPR-Cas9 genome editing, should enable engineering of K. marxianus isolates with promising lipid production at temperatures far exceeding those of other fungi under development for industrial applications. These results establish K. marxianus as a synthetic biology platform comparable to S. cerevisiae, with naturally more robust traits that hold potential for the industrial production of renewable chemicals.

synthetic biology↗

bioRxiv2018

Cancer-associated mRNAs regulated by the Helix-Loop-Helix motif of human EIF3A

Improper regulation of translation initiation, a vital checkpoint of protein synthesis in the cell, has been linked to a number of cancers. Overexpression of protein subunits of eukaryotic translation initiation factor 3 (eIF3) is associated with increased translation of mRNAs involved in cell proliferation. In addition to playing a major role in general translation initiation by serving as a scaffold for the assembly of translation initiation complexes, eIF3 regulates translation of specific cellular mRNAs and viral RNAs. Mutations in the N-terminal Helix-Loop-Helix (HLH) RNA-binding motif of the EIF3A subunit interfere with Hepatitis C Virus Internal Ribosome Entry Site (IRES) mediated translation initiation in vitro. Here we show that the EIF3A HLH motif controls translation of a small set of cellular transcripts enriched in oncogenic mRNAs, including MYC. We demonstrate that the HLH motif of EIF3A acts specifically on the 5-UTR of MYC mRNA and modulates the function of EIF4A1 on select transcripts during translation initiation. In Ramos lymphoma cell lines, which are dependent on MYC overexpression, mutations in the HLH motif greatly reduce MYC expression, impede proliferation and sensitize cells to anti-cancer compounds. These results reveal the potential of the EIF3A HLH motif in eIF3 as a promising chemotherapeutic target.

cancer biology↗

bioRxiv2018

Orthogonal regulation of ferritin translation by human eIF3 and the iron response proteins

A central problem in human biology remains the discovery of causal molecular links between mutations identified in genome-wide association studies (GWAS) and their corresponding disease traits. This challenge is magnified for variants residing in non-coding regions of the genome. Single-nucleotide polymorphisms (SNPs) in the 5 untranslated region (5-UTR) of the ferritin light chain (FTL) gene that cause hyperferritinemia are thought to disrupt translation repression by altering iron regulatory protein (IRP) interactions with the FTL mRNA 5-UTR. Here, we show that human eukaryotic translation initiation factor 3 (eIF3) acts as a distinct repressor of FTL mRNA translation, and eIF3-mediated FTL repression is disrupted by a subset of SNPs in FTL that cause hyperferritinemia. These results identify a direct role for eIF3-mediated translational control in a specific human disease.

molecular biology↗

bioRxiv2018

Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule

Small molecules that target the ribosome generally have a global impact on protein synthesis. However, the drug-like molecule PF-06446846 (PF846) binds the human ribosome and selectively blocks the translation of a small subset of proteins by an unknown mechanism. In high-resolution cryo-electron microscopy (cryo-EM) structures of human ribosome nascent chain complexes stalled by PF846, PF846 binds in the ribosome exit tunnel in a newly-identified and eukaryotic-specific pocket formed by the 28S ribosomal RNA (rRNA), and redirects the path of the nascent polypeptide chain. PF846 arrests the translating ribosome in the rotated state that precedes mRNA and tRNA translocation, with peptidyl-tRNA occupying a mixture of A/A and hybrid A/P sites, in which the tRNA 3-CCA end is improperly docked in the peptidyl transferase center. Using mRNA libraries, selections of PF846-dependent translation elongation stalling sequences reveal sequence preferences near the peptidyl transferase center, and uncover a newly-identified mechanism by which PF846 selectively blocks translation termination. These results illuminate how a small molecule selectively stalls the translation of the human ribosome, and provides a structural foundation for developing small molecules that inhibit the production of proteins of therapeutic interest.

biophysics↗

bioRxiv2017

Programmable RNA recognition using a CRISPR-associated Argonaute

Argonaute proteins (Agos) are present in all domains of life. While the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci and use non-canonical 5-hydroxyled guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2'-deoxyuridine (BrdU) as the 5 nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute-gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5-BrdU modified gRNAs, we mapped the seed region of the gRNA, and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the 6th and 7th positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific Adenosine to Inosine edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5-BrdU modified gRNAs can be used as a highly-specific RNA-targeting platform to probe RNA biology.\n\nSIGNIFICANCEArgonaute proteins are present in bacteria, archaea and eukaryotes. They play an important role in a wide range of biological processes, from transcriptional and translational gene expression regulation to defense against viruses and silencing of mobile genetic elements. Here we present mechanistic insights into the interactions of the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) with its guide RNA (gRNA) and RNA substrates. By modifying the 5-nucleotide of the gRNA, we demonstrate that MpAgo-gRNA complexes (RNPs) are easily programmable, have high affinity to fully complementary RNA substrates, and can discriminate by over 300 fold between substrates that differ by only a single nucleotide. These MpAgo RNPs should be useful for probing endogenous RNAs in living cells.

biochemistry↗

bioRxiv2017

Role for Ribosome-Associated Complex and Stress-Seventy subfamily B (RAC-Ssb) in integral membrane protein translation

Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle (SRP), which recognizes a hydrophobic signal sequence near the protein N-terminus. Proper folding of these proteins is monitored by the unfolded protein response, and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix-the signal sequence recognized by SRP-is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex (RAC) and Stress-Seventy Subfamily B (Ssb) chaperones. We also show that quality control of integral membrane proteins by RAC-Ssb couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology.

molecular biology↗

bioRxiv2016

A compound that directly and selectively stalls PCSK9 translation

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) plays a key role in regulating the levels of plasma low density lipoprotein cholesterol (LDL-C). Here we demonstrate that the compound PF-06446846 inhibits translation of PCSK9 by inducing the ribosome to stall around codon 34, mediated by the sequence of the nascent chain within the exit tunnel. We further show that PF-06446846 reduces plasma PCSK9 and total cholesterol levels in rats following oral dosing. Using ribosome profiling, we demonstrate that PF-06446846 is highly selective for the inhibition of PCSK9 translation. The mechanism of action employed by PF-06446846 reveals a previously unexpected tunability of the human ribosome, which allows small molecules to specifically block translation of individual transcripts.\n\nOne Sentence SummaryA small-molecule PCSK9 inhibitor targets the human ribosome and selectively prevents PCSK9 synthesis.

systems biology↗