Bro1 coordinates deubiquitination in the multivesicular body pathway by recruiting Doa4 to endosomes

Ubiquitination directs the sorting of cell surface receptors and other integral membrane proteins into the multivesicular body (MVB) pathway. Cargo proteins are subsequently deubiquitinated before their enclosure within MVB vesicles. In Saccharomyces cerevisiae, Bro1 functions at a late step of MVB sorting and is required for cargo protein deubiquitination. We show that the loss of Bro1 function is suppressed by the overexpression of DOA4, which encodes the ubiquitin thiolesterase required for the removal of ubiquitin from MVB cargoes. Overexpression of DOA4 restores cargo protein deubiquitination and sorting via the MVB pathway and reverses the abnormal endosomal morphology typical of bro1 mutant cells, resulting in the restoration of multivesicular endosomes. We further demonstrate that Doa4 interacts with Bro1 on endosomal membranes and that the recruitment of Doa4 to endosomes requires Bro1. Thus, our results point to a key role for Bro1 in coordinating the timing and location of deubiquitination by Doa4 in the MVB pathway

LSM1 over-expression in Saccharomyces cerevisiae depletes U6 snRNA levels

Lsm1 is a component of the Lsm1-7 complex involved in cytoplasmic mRNA degradation. Lsm1 is over-expressed in multiple tumor types, including over 80% of pancreatic tumors, and increased levels of Lsm1 protein have been shown to induce carcinogenic effects. Therefore, understanding the perturbations in cell process due to increased Lsm1 protein may help to identify possible therapeutics targeting tumors over-expressing Lsm1. Herein, we show that LSM1 over-expression in the yeast Saccharomyces cerevisiae inhibits growth primarily due to U6 snRNA depletion, thereby altering pre-mRNA splicing. The decrease in U6 snRNA levels causes yeast strains over-expressing Lsm1 to be hypersensitive to loss of other proteins required for production or function of the U6 snRNA, supporting a model wherein excess Lsm1 reduces the availability of the Lsm2-7 proteins, which also assemble with Lsm8 to form a complex that binds and stabilizes the U6 snRNA. Yeast strains over-expressing Lsm1 also display minor alterations in mRNA decay and demonstrate increased susceptibility to mutations inhibiting cytoplasmic deadenylation, a process required for both 5′-to-3′ and 3′-to-5′ pathways of exonucleolytic decay. These results suggest that inhibition of splicing and/or deadenylation may be effective therapies for Lsm1-over-expressing tumors

LSM1 AND RNY1: CLUES IN THE SEARCH FOR HOW RNA METABOLIC PATHWAYS CONTROL CANCER

Carcinogenesis requires numerous alterations to gene expression to evade normal controls on cellular growth, invasion, and immortality. Traditionally, these changes have been examined in the context of deregulated transcriptional control of oncogenes and tumor suppressors. However, in recent years, research has revealed that processes outside of transcription such as RNA splicing, translation, and decay are also deregulated in cancers, sustaining tumorigenic potential. This dissertation details our investigation into the cellular functions of two RNA metabolic proteins whose human orthologs are deregulated in tumors: a putative oncogene, Lsm1, and a putative tumor suppressor, Rny1. Herein, we reveal interesting functions for these proteins that might provide insight into their roles in carcinogenesis. First, we demonstrate a role for Lsm1 over-expression in altered splicing through depletion of U6 snRNA levels. Second, we clarify the mechanism for Rny1's activity against RNA substrates and identify cis regions required for its non-catalytic role in growth inhibition. Overall, this knowledge expands our understanding of how RNA metabolism might be deregulated in cancer and could provide novel pathways to target for synthetic lethal responses in cancers with altered expression of these proteins.Embargo: Release after 4/7/201

Structure-function analysis of Rny1 in tRNA cleavage and growth inhibition.

T2 ribonucleases are conserved nucleases that affect a variety of processes in eukaryotic cells including the regulation of self-incompatibility by S-RNases in plants, modulation of host immune cell responses by viral and schistosome T2 enzymes, and neurological development and tumor progression in humans. These roles for RNaseT2's can be due to catalytic or catalytic-independent functions of the molecule. Despite this broad importance, the features of RNaseT2 proteins that modulate catalytic and catalytic-independent functions are poorly understood. Herein, we analyze the features of Rny1 in Saccharomyces cerevisiae to determine the requirements for cleaving tRNA in vivo and for inhibiting cellular growth in a catalytic-independent manner. We demonstrate that catalytic-independent inhibition of growth is a combinatorial property of the protein and is affected by a fungal-specific C-terminal extension, the conserved catalytic core, and the presence of a signal peptide. Catalytic functions of Rny1 are independent of the C-terminal extension, are affected by many mutations in the catalytic core, and also require a signal peptide. Biochemical flotation assays reveal that in rny1Δ cells, some tRNA molecules associate with membranes suggesting that cleavage of tRNAs by Rny1 can involve either tRNA association with, or uptake into, membrane compartments

Sequence and structural analysis of Rny1.

<p>(A) Diagram indicating the positions within the amino acid sequence of <i>RNY1</i> regions analyzed by deletion. (B) COBALT alignment (<a href="http://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi" target="_blank">http://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi</a>) of Rny1 of <i>S. cerevisiae</i> (top, in blue) to other ribonucleases of known structure (Rh, <i>R. niveus</i>, middle, in red; ACTIBIND, <i>A. niger</i>, bottom, in khaki) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041111#pone.0041111-deLeeuw1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041111#pone.0041111-Kurihara1" target="_blank">[32]</a>. T2 ribonuclease conserved amino acid sequences (CAS) are underlined and shown in red (I) and light red (II). Predicted nucleotide binding residues are shown in blue (B1 site) and yellow (B2 site) and are based on an alignment of Rh to ribonucleases whose structures are known in complex with nucleotides <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041111#pone.0041111-Rodriguez1" target="_blank">[33]</a>. Residues that overlap involvement in B1 and CAS are shown in purple while those that overlap B2 and CAS are designated by orange (conserved sequence elements of T2 ribonucleases are reviewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041111#pone.0041111-Luhtala1" target="_blank">[13]</a>). Putative N-linked glycosylation sites are depicted by underlined pink N residues which were identified by analysis with predictive glycosylation software (<a href="http://comp.chem.nottingham.ac.uk/cgi-bin/glyco/bin/getparams.cgi" target="_blank">http://comp.chem.nottingham.ac.uk/cgi-bin/glyco/bin/getparams.cgi</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041111#pone.0041111-Caragea1" target="_blank">[37]</a>). Loops targeted for mutation are boxed in pink and labelled L1–L10, corresponding to the structure in (C). Green and red circles above boxes indicate whether these loops were tested in active and/or inactive Rny1 backgrounds, respectively, with results of these analyses in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041111#pone-0041111-t001" target="_blank">Table 1</a>. (C) Swiss Model predicted structure (Swiss Model (<a href="http://swissmodel.expasy.org/" target="_blank">http://swissmodel.expasy.org/</a>) was generated by 39% homology to ACTIBIND (de Leeuw, Roiz et al. 2007), and the image was illustrated in cyan using PyMol (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>) with color coding and loop designations referring to those used in (A). Catalytic histidine residues are shown as protrusions within the T2 core in orange and purple. Loops L4 and L7 are predicted to participate in nucleotide binding based on our alignment to Rh which was previously aligned to ribonucleases with known regions of nucleotide binding (Rodriguez, 2008 #476).</p

The signal peptide and T2 region affect tRNA cleavage.

<p>(A) Northern blot performed, blotting for tRNA Met(CAT), as detailed in Materials and Methods. Strains deleted for <i>RNY1</i> expressing <i>GAL-RNY1</i> mutant constructs (abbreviations defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041111#pone-0041111-g002" target="_blank">Figure 2</a>) expressed in the catalytically active background. Migration of oligonucleotide standards is shown in base pairs (bp). (B) Western blot (performed as indicated in Materials and Methods) of strains expressing constructs as shown in (A). Migration of molecular weight standards is indicated.</p

Multiple regions of Rny1 affect growth.

<p>(A) Frog ponds (performed as described in Materials and Methods section) on synthetic selective media plates containing galactose to induce Rny1’s over-expression in catalytic mutant background in a <i>hir2</i>Δ strain as a <i>GAL-RNY1</i> plasmid either full-length (WT), deleted for either the signal peptide sequence (ΔSP), the T2 conserved region (ΔT2) or the unique C-terminal region (ΔCTD) or a vector control (v). (B) Western blot (performed as indicated in Materials and Methods) of strains expressing constructs as shown in (A) except that the first lane shows a non-catalytic, full-length mutant GAL-RNY1’s expression in the same strain (WT). Migration of molecular weight standards is indicated.</p

Rny1 cleaves tRNA at vacuoles.

<p>Equal amounts of cells were used to make dextran-treated spheroplasts of <i>rny1</i>Δ expressing Rny1 on a low-copy plasmid (Rny1–13myc) or vector (vec), prepared after one day of growth from midlog. These were floated on Ficoll gradients (0%, 4%, 8%, and 12% Ficoll solutions layered over samples in 15% Ficoll) to probe for Rny1’s impact on RNA cleavage at vacuoles, process is diagrammed in (A). Even-numbered fractions examined on Western blots and Northern blots. (B) <i>upper panel</i>. RNA from equal volumes of Ficoll fractions resolved by urea-acrylamide electrophoresis and Northern blotted for tRNA His (GTG). Input lanes were loaded at 50%. Position of fragments is indicated. Migration of oligonucleotide standards is shown in base pairs (bp). Numbers given above lanes represent fraction numbers on Ficoll gradients from top to bottom as diagrammed in (A) All samples are from the same image of the same blot, and the image was cropped after scaling the image to show only relevant samples. <i>lower panel</i>. Equal volumes of Ficoll fractions from the same experiment were denatured in buffer and resolved by SDS-PAGE. Input lanes were loaded at 50%. Western blots were performed for the indicated proteins to represent vacuoles (CPY, carboxypeptidase Y), ER (Dpm1), and mitochondria (Porin). Migration of molecular weight standards is indicated.</p

A glycosylation mutant lacks RNA cleavage activity.

<p>(A) Northern blot probing for tRNA Met(CAT). Strains deleted for <i>RNY1</i> expressing Rny1-GFP, GFP-Rny1, or vector. Migration of oligonucleotide standards is shown in base pairs (bp). (B) PNGase F or control digests of total lysates of wild-type strains expressing Rny1-GFP or GFP-Rny1. Samples were resolved by SDS-PAGE and probed by Western blot for GFP. Migration of molecular weight standards is indicated.</p

Multiple loops regulate RNA cleavage.

<p>(A) Cleavage of tRNA Met(CAT) by over-expressed full-length (WT), full-length catalytically inactive (ci), vector control (v), or RNY1 containing catalytic histidines but mutated in <i>cis</i> at indicated loops (L#). Strains deleted for <i>RNY1</i> were grown as described to induce plasmid over-expression of <i>GAL-RNY1</i> in the plasmid mutant or control indicated, and equivalent amounts of RNA were resolved and transferred to gels for Northern blots using oRP1401, all performed as indicated in the Materials and Methods. All samples are from the same image of the same blot, and the image was cropped after scaling the image to show only relevant samples (for A and B). Bracket reveals where expected bands accumulate with over-expression of the full-length (WT) <i>RNY1</i>. Migration of oligonucleotide standards is shown in base pairs (bp). (B) Western blot (performed as indicated in Materials and Methods) of strains expressing constructs as shown in (A). Migration of molecular weight standards is indicated.</p