Browsing by Subject "Ribosome"

In mammals, the perception of smell starts with the activation of odorant receptors (ORs) by volatile molecules in the environment. Mammalian genomes typically encode large numbers of ORs, with approximately 400 intact ORs in human and more than 1000 in mouse. Central to the question of how olfactory stimuli are represented at the peripheral level is defining the ligand selectivity and activity regulation of ORs.

Processing of chemosensory signals in the brain is dynamically regulated in part by an animal’s physiological state. The Matsunami lab previously reported that type 3 muscarinic acetylcholine receptors (M3-Rs) physically interact with odorant receptors (ORs) to promote odor-induced responses in a heterologous expression system. However, it is not known how M3-Rs affect the ability of olfactory sensory neurons (OSNs) to respond to odors. In chapter 2, I demonstrate that the activation of M3-Rs inhibits the recruitment of β-arrestin-2 to ORs, resulting in a potentiation of odor-induced response in OSNs. These results suggest a role for acetylcholine in modulating olfactory processing at the initial stages of signal transduction in the olfactory system.

Understanding odor coding requires comprehensive mapping between odorant receptors and corresponding odorants. In chapter 3, I present a high-throughput in vivo method to identify repertoires of odorant receptors activated by odorants, using phosphorylated ribosome immunoprecipitation of mRNA from olfactory epithelium of odor-stimulated mice followed by RNA-Seq. This approach screens endogenously expressed odorant receptors against an odorant in one set of experiments, using awake and freely behaving mice. In combination with validations in a heterologous system, we identify sets of odorant receptors for two odorants, acetophenone and 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), encompassing 69 receptor-odorant pairs. I also identified shared amino acid residues specific to the acetophenone or TMT receptors, and developed a model to predict receptor activation. This study provides a means to understand the combinatorial coding of odors in vivo.

The signal recognition particle (SRP) pathway has long been regarded as the primary mechanism of transcriptome and translatome partitioning to the endoplasmic reticulum (ER). This co-translational targeting mechanism is conserved in all living organisms studied to date. By the SRP pathway, ribosomes translating secretory and membrane protein-encoding mRNAs in the cytosol are selected for recruitment to the ER following presentation of a peptide signal sequence early in translation. SRP recognizes and binds the peptide signal and targets the mRNA-ribosome-nascent chain complex to the ER membrane via interaction with the ER-resident SRP receptor (SR). The membrane-targeted signal peptide is then passed to the translocon and the secretory/membrane protein is co-translationally translocated into the ER lumen or inserted into the ER membrane. Via this positive selection strategy, mRNAs encoding secretory and membrane proteins are translated by ER-associated ribosomes, while non-signal encoding mRNAs are translated by free ribosomes in the cytosol. Yeast and bacterial species possess post-translational protein translocation mechanisms which allow SRP-independent translocation. Mammalian cells, by contrast, rely on the SRP pathway for protein targeting and translocation. The precise role of the SRP pathway in subcellular mRNA localization, however, remains elusive. In fact, studies which investigate RNA localization to the ER suggest many diverse strategies could anchor a broad representation of all cellular mRNAs to the ER membrane and regulate their translation. Here we evaluate the extent to which the SRP pathway contributes to transcriptome partitioning in mammalian cells via CRISPR/Cas9- and RNAi-mediated depletion of SR, followed by sequential detergent fractionation into cytosol and ER subcellular fractions and deep sequencing of the compartmentalized mRNAs. We found that disruption of the SRP pathway does not impact steady-state mRNA localization to the ER, though minor defects in protein expression were observed in a quantitative proteomic study, thereby decoupling SRP pathway function in protein biogenesis from general mRNA partitioning. Assessment of de novo subcellular localization patterns of newly synthesized mRNAs, via deep sequencing of 4-thiouridine metabolically labeled mRNAs in the cytosol and ER fractions of parental and SR-deficient cells, revealed that mRNAs are predominately localized to the ER membrane upon nuclear export, independently of a functional SRP pathway or encoded signal sequence. We further found that translation inhibition, through physiological stress or chemical inhibitors, enhanced the ER localization of mRNAs, especially non-signal encoding mRNAs. This suggests that translation-coupled events release this mRNA cohort into the cytosol to establish steady-state subcellular distributions. Additional investigation into RNA localization patterns by single molecule RNA imaging under conditions of stress-induced translation inhibition, which promotes the formation of ribonucleoprotein stress granules (SG), revealed that newly synthesized mRNAs serve as primary substrates for SG biogenesis, as transcription inhibition prevented mRNA recruitment into SGs. Furthermore, SG formation was found to occur in association with the ER membrane for both signal- and non-signal-encoding mRNAs. Collectively these data support a novel mRNA trafficking model by which newly synthesized mRNAs are exported from the nucleus and localized directly to the ER membrane independently of the SRP pathway, likely via interaction with ER-resident ribosome and/or RNA binding proteins, implicating the ER as a regulatory center for initiation of subcellar transcriptome partitioning.

RNA is the molecular workhorse of nature, capable of doing many cellular tasks, from genetic data storage and regulation, to enzymatic synthesis--even to the point of self-catalyzing its own replication. While RNA can act as a catalyst on its own, as in the hammerhead ribozyme, the added efficiency of proteins is often a necessity; the ribosome--the large ribozyme responsible for peptide chain formation, is aided by proteins which ensure correct assembly and structural stability. These complexes of RNA and proteins feature in many essential cellular processes, including the RISC silencing complex and in the spliceosome. Despite its enormous utility, structural determination of RNA is notoriously difficult--particularly in the backbone, since a nucleotide standardly has 12 torsion angles (including χ) and 12 non-hydrogen atoms, compared to 4 torsions (including χ1) and 4 non-H atoms in a typical amino acid. The abundance of backbone atoms, their conformational flexibility, and experimental resolution limitations often result in systematic errors that can have a significant impact on the interpretation. False trails due to structural errors can lead to significant loss of time and effort, especially with such high-profile complexes as the ribosome and the RISC complex.

My research has focused on harnessing the recently discovered ribosome structures and the Richardsons' RNA dataset to find trends in RNA backbone conformations and motifs that were then used to develop structural validation techniques and provide improved diagnosis and correction techniques for RNA backbone. Methods for fixing RNA structure have been developed for both NMR and X-ray crystallography. For NMR structures, a method for assigning RNA backbone structure based on NOE data was developed, leading to improved identification and building of RNA backbone conformation in NMR ensembles. For crystallography, our method of diagnosing the correct ribose pucker from clear observables allows reliable assessment of pucker in validation or refinement. Observed differences in bond-lengths, bond-angles, and dihedrals have been categorized by sugar pucker in the PHENIX refinement package. I have shown that this improves the refinement behavior of both pucker and geometry.

There have also been improvements in identifying structural motifs. Many previously identified structural motifs have now been defined in terms of backbone suitestrings, a series of 2-character code divisions of RNA backbone that show the best clustering of dihedral angle correlations. Combined with a BLAST-like alignment program called SuiteAlign, these suitestrings were quickly and easily identified in a number of structures, eventually leading to the discovery of multiple instances of TψC-loop structures in the ribosome.

To facilitate error diagnosis and corrections in RNA-protein complexes, as well as to expand the knowledge base of the scientific community as a whole, a database of RNA-protein interaction motifs has been developed. This database is rooted in the quality-filtering, visualization, and analysis techniques of the Richardson lab, particularly those developed by Laura Murray specifically for RNA structures.

The consensus backbone conformers, pucker diagnosis, and all-atom contacts have been combined to develop first manual and then automated tools for RNA structure correction. I have applied all these techniques to improve the accuracy of a number of important RNA and RNA/protein complex structures.

The partitioning of translation to the outer membrane of the endoplasmic reticulum is a problem that has been the subject of inquiry since the discovery of the ribosome. The large degree to which ribosomes were found to be tethered to the membrane led to intense investigation of a series of related questions regarding the identity of those mRNAs that are translated on the endoplasmic reticulum, and the functions of that localization in cell stress. In this dissertation, I approach each of these questions in turn and work to reconcile my observations with those models that have been previously proposed. A theme of this work is the application of modern methods, particularly deep sequencing technology, to address problems that had largely been considered solved. The most prominently featured method is ribosome profiling, which is paired with classical biochemical and cell biological techniques. I arrive at several conclusions: 1) a significant fraction of all mRNAs is well represented on the endoplasmic reticulum membrane, 2) the properties of translation diverge substantially between membrane-associated and free ribosomes, and 3) the compartmentalization of translation can serve as an important variable in cell stress.

While much is known about the abundance and genetic diversity of environmental microbial communities, little is known about their taxon-specific activity. In this thesis I address this gap using a model marine microbe, the cyanobacterium Prochlorococcus spp., which numerically dominates tropical and subtropical open oceans and encompasses a group of genetically defined clades that are ecologically distinct. Ribosomal RNA is a promising indicator of in situ activity because of its essential role in protein synthesis as well as its phylogenetic information, which could be used to distinguish clades among mixed populations. Here I show that, in a laboratory system the specific growth rate of representative Prochlorococcus strains could be quantitative predicted from cellular rRNA content (assessed by RT-qPCR), cell size (assessed by flow cytometry) and temperature. Applying this approach in the field, I show unique clade-specific activity patterns for Prochlorococcus. For example, vertically within the euphotic zone, eHL-II activity is strongly impacted by light and is consistent with patterns of photosynthesis and on a horizontal transect from Hawaii to San Diego, eHL-I and eHL-II activities exhibit significant transitions and appear to be regulated by temperature, nutrient and vertical mixing gradients. Using ribosomal tag pyrosequencing of DNA and RNA, I have extended our observation to the Eubacterial community and described the biomass distribution (rDNA) and activity (rRNA) patterns from two representative depths (25 and 100 m) at a well-studied oligotrophic ocean station. These results show that for some populations the abundances and activities are significantly uncoupled, which suggests substantial top-down controls or physical transport processes. Further exploring the taxon-specific activity patterns along with abundances and environmental variables across time and space is essential to better understanding the dynamics of a complex microbial system as well as predicting the consequences of environmental change.

The goal of this research is to identify the trafficking patterns that direct ribosomes to the endoplasmic reticulum (ER). It is widely believed that the SRP pathway is the only mechanism that cells use to localize mRNA and ribosomes to the ER, but this has been found not to be a sufficient explanation for the patterns of RNA localization in cells, namely that non-signal sequence-containing mRNA are translated on the ER and that ribosomes retain their membrane association after translation termination. First, a summary of the history of the field is presented to provide context for the key, unanswered questions in the field. Then, experiments employing [32Pi] pulse-chase labeling of HeLa cells over a time course to follow nascent ribosome trafficking are presented. The purpose of the cell labeling was to track rRNA processing and assembly into nascent ribosomes, followed by their export into the cytoplasm and recruitment into active polysomes. A detergent-based cell fractionation procedure was also utilized to separate the cytosol and ER compartments in order to observe ribosomes on their path as they exit the nucleus and either localize to the ER or cytosolic cellular compartment. Through this method, it was seen that ribosomes appear in both compartments at the same time, suggesting a mechanism may be occurring in addition to SRP-dependent ribosome trafficking. This research provides an understanding toward a mechanism that is not currently known, but will one day more fully explain the patterns of ribosomal localization.