PRM-based quantitative proteomic analysis of mouse tissues, testicular cells and cell lines
ABSTRACT: PRM analysis of RPL39L, RPL10L and RPL3L in 9 mouse tissues, including testis, fat, brain, liver, kidney, lung, skeletal muscle, heart and spleen; PRM analysis of RPL39 and RPL39L, RPL10 and RPL10L, and RPL22 and RPL22L1 in testicular cells; PRM analysis of RPL39L and RPL39 in RibosomeRPL39L-/- and wild type mouse spermatocytes and spermatids; PRM analysis of 17 down-regulated proteins and 1 unchanged protein (DYNC1LI2) in RibosomeRPL39L-/- testes; PRM analysis of RPL39 and RPL39L in GC-1 ribosomes, and RibosomeRPL39L-/- and wild type N2a ribosomes, Consistency between the observed and expected signal ratios of the light and heavy peptides by PRM in serial dilution experiments. Bovine serum albumin (BSA) as a negative control.
Project description:Ribosomes are essential macromolecular complexes conducting protein biosynthesis in all domains of life. Cells can have heterogeneous ribosomes, i.e. ribosomes with various ribosomal RNA (rRNA) and ribosomal protein (r-protein) composition. However, the functional importance of heterogeneous ribosomes has remained elusive. One of the possible sources for ribosome heterogeneity is provided by paralogous r-proteins. In E. coli, ribosomal protein bL31 has two paralogs: bL31A encoded by rpmE and bL31B encoded by ykgM. This study investigates phenotypic effects of these ribosomal protein paralogs using bacterial strains expressing only bL31A or bL31B. We show that bL31A confers higher fitness to E. coli under lower temperatures. In addition, bL31A and bL31B have different effects on translation reading frame maintenance and apparent translation processivity in vivo as demonstrated by dual luciferase assay. In general, this study demonstrates that ribosomal protein paralog composition (bL31A versus bL31B) can affect cell growth and translation outcome.
Project description:Cotranslational targeting into the endoplasmic reticulum (ER) by the Signal Recognition Particle (SRP) is a key event determining polypeptide fate in eukaryotic cells. Here, we globally define the principles and mechanisms of SRP binding and ER targeting in vivo. Cotranslational targeting through SRP is the dominant route into the ER for all secretory proteins, regardless of targeting signal characteristics. Cytosolic SRP functions in a pioneer translation round that builds a membrane-resident mRNAs pool, explaining how low SRP levels suffice for the secretory load. SRP does not induce an elongation arrest; consequently, kinetic competition between targeting and translation elongation dictates which substrates are translocated post-translationally. Unexpectedly, SRP binds most secretory ribosomal complexes before targeting signals are synthesized. We show non-coding mRNA elements can promote signal-independent SRP pre-recruitment. Our study defines the complex kinetic interplay between elongation and determinants in the polypeptide and mRNA modulating SRP-substrate selection and membrane targeting in vivo. Ribosome profiling (RiboSeq) and RNA-seq of subcellular fractions of ribosomes. Soluble and membrane bound ribosomes are separated by centrifugation, and SRP-bound ribosomes are immunoprecipitated from the soluble fraction. Polysomes and monosomes are separated by sucrose gradient ultracentrifugation.
Project description:Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5 - 7 days to generate a completed ribosome profiling sequencing library. Ribosome profiling in cultured mammalian cells under three different footprinting conditions
Project description:Ribosomes are produced in large quantities during oogenesis and stored in the egg. However, the egg and early embryo are translationally inactive. How translation is activated during embryogenesis is poorly understood. Using mass-spectrometry and cryo-EM analyses of ribosomes isolated from zebrafish and Xenopus eggs and embryos, we find that ribosomes transition from a dormant to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four factors that form two modules: a Habp4-eEF2 module that stabilizes ribosome levels and a Dap1b/Dapl1-eIF-5a module that represses translation. Dap/Dap1b is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Thus, a developmentally programmed, unique ribosome state plays a key role in ribosome storage and translational repression in the egg.
Project description:The 120-nt long 5S rRNA, is an indispensable component of cytoplasmic ribosomes in all living organisms. The functions of 5S rRNA and the reasons for its evolutionary preservation as an independent molecule remain unclear. Here we used ribosome engineering to investigate whether maintaining 5S rRNA as an independent molecule is critical for ribosome function and cell survival. By fusing circularly permutated 5S rRNA (cp5S) with 23S rRNA and deleting all wild type 5S rRNA genes, we generated an Escherichia coli strain completely devoid of free 5S rRNA. Viability of the engineered cells demonstrates that autonomous 5S rRNA is not required for cell growth at 37°C and is unlikely to have essential functions outside the ribosome. The fully-assembled ribosomes carrying 23S-cp5S hybrid rRNA and lacking free 5S rRNA are highly active in translation. However, the engineered cells accumulate aberrant 50S subunits that are unable to form stable 70S ribosomes. Cryo-EM analysis revealed a dramatically malformed peptidyl transferase center in the misassembled 50S subunits. The results of our experiments argue that the key evolutionary force preserving the autonomous nature of the smallest rRNA is its role in ribosome biogenesis.
Project description:Translational regulation can be studied on a global scale by integrating polysome fractionation of mRNAs with microarray hybridization. This approach is based on the fact that translationally quiescent mRNAs are sequestered within messenger ribonucleoprotein (mRNP) particles or associated with single ribosomes (monosomes), whereas actively translated mRNAs are associated with multiple ribosomes (polysomes). The mRNAs associated within these fractions are then used to interrogate microarrays, providing insight into how the translational state of individual mRNAs is modified by environmental cues. In this study, we coupled polysome fractionation with microarray detection in order to identify changes in the translation state of the A. fumigatus transcriptome under conditions that perturb ER homeostasis such as chemical stress (DTT, tunicamycin) or thermal stress (shift from 25 degrees celsius to 37 degrees celsius).
Project description:DNA-repair factors of the Nucleotide -Excision-Repair (NER) pathway are part of the basal transcription apparatus of RNA polymerase I. Mutations in these factors can give rise to developmental disorders with symptoms that are typical for the aging body. Here we show that in Cockayne syndrome (CS) RNA polymerase I transcription elongation and the processing of the pre-rRNA are disturbed. The mature 18S rRNA is reduced and isolated ribosomes lack specific ribosomal proteins. Ribosomal proteins are susceptible to unfolding and the proteome of CS cells is heat-sensitive, indicating misfolded proteins and an error prone translation process in CS cells. Pharmaceutical chaperones restored impaired cellular proliferation. Thus, we provide evidence for severe malfunction in protein synthesis which together with a loss of proteostasis constitute the underlying pathophysiology in CS.
Project description:Hypoxic conditions prompt internal ribosome entry site (IRES)-mediated translation of some of the hallmark cancer genes, such as VEGF. This translational switch is extremely vital for cell survival and tumor progression. Heterogeneity in ribosomes due to the diversity of ribosomal RNA (rRNA) and protein composition has been postulated to generate ‘specialized ribosomes’ that differentially regulate translation. A VEGF IRES sequence was used as bait to identify unique proteins bound at the IRES in breast cancer cells grown in hypoxia.
Project description:The interactions between RNA polymerase and ribosomes are critical for the coordination of transcription with translation. We report that RNA polymerase directly binds ribosomes and isolated large and small ribosomal subunits.