3.88 A structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy.

Cytoplasmic polyhedrosis virus (CPV) is unique within the Reoviridae family in having a turreted single-layer capsid contained within polyhedrin inclusion bodies, yet being fully capable of cell entry and endogenous RNA transcription. Biochemical data have shown that the amino-terminal 79 residues of the CPV turret protein (TP) is sufficient to bring CPV or engineered proteins into the polyhedrin matrix for micro-encapsulation. Here we report the three-dimensional structure of CPV at 3.88 A resolution using single-particle cryo-electron microscopy. Our map clearly shows the turns and deep grooves of alpha-helices, the strand separation in beta-sheets, and densities for loops and many bulky side chains; thus permitting atomic model-building effort from cryo-electron microscopy maps. We observed a helix-to-beta-hairpin conformational change between the two conformational states of the capsid shell protein in the region directly interacting with genomic RNA. We have also discovered a messenger RNA release hole coupled with the mRNA capping machinery unique to CPV. Furthermore, we have identified the polyhedrin-binding domain, a structure that has potential in nanobiotechnology applications.

Mechanisms of protein regulation in rat skeletal muscle during resistance exercise and training

The cellular mechanisms through which adult rat skeletal muscle protein is regulated during resistance exercise and training was investigated. A model of non-voluntary resistance exercise was described which involves the electrically-stimulated contraction of the lower leg muscles of anesthetized rats against a weighted pulley-bar. Muscle protein synthesis rates were measured by in vivo constant infusion of $\sp3$H-leucine following a single bout of resistance exercise. Specific messenger RNA levels were determined by dot-blot hybridization analysis using $\sp{32}$P-labelled DNA probes after a single bout and multiple bouts of phasic training. The effects of phasic training on increasing skeletal muscle mass was assessed. Between 12 and 36 hours following a single resistance exercise bout (24-192 contractions), total mixed and myofibril protein synthesis rates were significantly increase (32%-65%) after concentric (gastrocnemius m.) and eccentric (tibialis anterior m.) contractions. Eccentric contractions had greater effects on myofibril synthesis with more prolonged increases in synthesis rates. Lower numbers of eccentric than concentric contractions were required to increase synthesis. Cellular RNA was increased after exercise but the relative levels of skeletal $\alpha$-actin and cytochrome c mRNAs were unchanged. Since increases in synthesis rates exceeded increases in RNA, post-transcriptional mechanisms may be primarily responsible for increased protein synthesis after a resistance exercise bout. After 10-22 weeks of phasic eccentric resistance training, muscle enlargement (16%-30%) was produced in the tibialis anterior m. after all training paradigms examined. In contrast, gastrocnemius m. enlargement after phasic concentric training occurred after moderate (24/bout) but not after high (192/bout) repetition training. The absence of muscle growth in the gastrocnemius m. after high repetition training despite increased synthesis rates after the initial bout and RNA and possibly mRNA accumulation during training suggests a role for post-translational mechanisms (protein degradation) in the control of muscle growth in the gastrocnemius m. It is concluded that muscle protein during resistance exercise and training is regulated at several cellular levels. The particular response may be influenced by the exercise intensity and duration, the training frequency and the type of contractile work (eccentric vs. concentric) performed. ^

Mapping and cloning of genes from portions of the human genome using somatic cell hybrids

Human x rodent somatic cell hybrids have played an important role in human genetics research. They have been especially useful for assigning genes to chromosomes and isolating DNA markers from specific regions of the human genome.^ By employing a combination of somatic cell genetic, recombinant DNA, and cytogenetic techniques, human DNA excision repair gene ERCC4 was mapped regionally to human 16p13.13-13.2, even though the gene has not been cloned. Human x Chinese hamster ovary (CHO) cell hybrids selected for human ERCC4 activity and containing 16p13.1-p13.3 as the only human genetic material were identified. These hybrids were used to order DNA markers located in 16p13.1-p13.3. New DNA markers physically close to ERCC4 were isolated from such hybrids. Using amplified human DNA from the hybrids as probe in fluorescent in situ hybridization, the short arm breakpoint in the chromosome 16 inversion associated with acute myelomonocytic leukemia (AMML) was found to be physically close to the ERCC4 gene. The physical mapping and eventually, the cloning of the ERCC4 gene, will benefit the understanding of the DNA repair system and the study of other important biomedical problems such as tumorigenesis.^ To facilitate the cloning of ERCC4 gene and, in general, the cloning of genes from any defined regions of the human genome, a method was developed for the direct isolation of human transcribed genes ffom somatic cell hybrids. cDNA was prepared from human x rodent hybrid by using consensus 5$\sp\prime$ splice site sequences as primers. These primers were designed to select immature, unspliced messenger RNA (still retaining species specific repeat sequences) as templates. Screening of a derived cDNA library for human repeat sequences resulted in the isolation of human clones at the anticipated frequency with characteristics expected of exons of transcribed human genes. The usefulness of the splice site specific primers was analyzed and the cDNA synthesis conditions with these primers were optimized. The procedure was shown to be sensitive enough to clone weakly expressed genes. Studying the expression of the represented genes with the isolated clones was shown to be feasible. Such regional specific human gene fragments will be very valuable for many human genetic studies such as the search of inherited disease genes and the construction of a cDNA map of the human genome. ^

GLUCAGON GENE EXPRESSION: ANALYSIS OF PREPROGLUCAGON

Glucagon is a 29 amino acid polypeptide hormone produced in the (alpha) cells of the pancreatic islets. The purpose of this research was to understand better the role of glucagon in the regulation of metabolic processes. As with other polypeptide hormones, the synthesis of glucagon is thought to involve a larger precursor, which is then enzymatically cleaved to the functional form. The specific research objectives were to obtain cloned copies of the messenger RNA (mRNA) for pancreatic glucagon, to determine their primary sequences, and from this coding information to deduce the amino acid sequence of the initial glucagon precursor. From this suggested preproglucagon sequence and prior information on possible proglucagon intermediate processing products, the overall objective of this research is to propose a possible pathway for the biosynthesis of pancreatic glucagon.^ Synthetic oligodeoxynucleotide probes of 14-nucleotides (14-mer) and 17-nucleotides (a 17-mer) complementary to codons specifying a unique sequence of mature glucagon were synthesized. The ('32)P-labeled-14-mer was hybridized with size-fractionated fetal bovine pancreatic poly(A('+))RNA bound to nitrocellulose. RNA fractions of (TURN)14S were found to hybridize specifically, resulting in an (TURN)10-fold enrichment for these sequences. These poly(A('+))RNAs were translated in a cell-free system and the products analyzed by gel electrophoresis. The translation products were found to be enriched for a protein of the putative size of mammalian preproglucagon ((TURN)21 kd). These enriched RNA fractions were used to construct a complementary DNA (cDNA) library is plasmid pBR322.^ Screening of duplicate colony filters with the ('32)P-labeled-17-mer and a ('32)P-labeled-17-mer-primed cDNA probe indicated 25 possible glucagon clones from 3100 colonies screened. Restriction mapping of 6 of these clones suggested that they represented a single mRNA species. Primary sequence analysis of one clone containing a 1200 base pair DNA insert revealed that it contained essentially a full-length copy of glucagon cDNA.^ Analaysis of the cDNA suggested that it encoded an initial translation product of 180 amino acids with an M(,r) = 21 kd. The first initiation codon (ATG, methionine) followed by the longest open reading frame of 540 nucleotides was preceded by a 5'-untranslated region of 90 nucleotides, and was followed by a longer 3'-untranslated region of 471 nucleotides, resulting in a total of 1101 nucleotides. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Mechanisms by which nonsense codons downregulate T-cell receptor transcripts

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that degrades aberrant mRNAs harboring premature termination codons (PTCs). Two out of three T-cell receptor β (TCRβ) transcripts carry PTCs as a result of error-prone programmed rearrangements that occur at this locus during lymphocyte maturation. PTCs decrease TCRβ mRNA levels to a much greater extent than mRNAs transcribed from non-rearranging genes. This robust decrease in TCRβ mRNA levels is not a unique characteristic of the T-cell environment or the TCRβ promoter. The simplest explanation for this is that PTC-bearing TCRβ mRNAs elicit a stronger NMD response. An alternative explanation is NMD collaborates with another mechanism to dramatically decrease PTC-bearing TCRβ mRNA levels. ^ In my dissertation, I investigated the molecular mechanism behind the strong decrease in TCRβ mRNA levels triggered by PTCs. To determine the location of this response, I performed mRNA half-life analysis and found that PTCs elicited more rapid TCRβ mRNA decay in the nuclear fraction, not the cytoplasmic fraction. Although decay was restricted to the nuclear fraction, PTC-bearing TCRβ transcript levels were extremely low in the cytoplasm, a phenomenon that I named the nonsense-codon induced partitioning shift (NIPS). I established that NIPS shares several qualities with NMD, including its dependence on translation and NMD factors. Several lines of evidence suggested that NIPS results from PTCs eliciting retention of TCRβ transcripts in the nuclear fraction. This retention, as well as rapid TCRβ mRNA decay, most likely occurs in either the nucleoplasm or the outer nuclear membrane, based on analysis of nuclear and cytoplasmic markers in the highly purified nuclei I used for my studies. To further address the location of decay, I asked whether nuclear or cytoplasmic RNA decay factors mediated the destruction of PTC-bearing mRNAs. My results suggested that a nuclear component of the 3'-to-5' exosome, as well as an endonucleolytic activity, are involved in the destruction of PTC-containing TCRβ mRNAs. Individual endogenous NMD substrates had differential requirements for nuclear and cytoplasmic exonucleases. In summary, my results provide evidence that PTCs trigger multiple mechanisms involving multiple decay factors to remove and regulate mRNAs in mammalian cells. ^

Modeling Techniques for the High-Resolution Interpretation of Cryo-Electron Microscopy Reconstructions

Essential biological processes are governed by organized, dynamic interactions between multiple biomolecular systems. Complexes are thus formed to enable the biological function and get dissembled as the process is completed. Examples of such processes include the translation of the messenger RNA into protein by the ribosome, the folding of proteins by chaperonins or the entry of viruses in host cells. Understanding these fundamental processes by characterizing the molecular mechanisms that enable then, would allow the (better) design of therapies and drugs. Such molecular mechanisms may be revealed trough the structural elucidation of the biomolecular assemblies at the core of these processes. Various experimental techniques may be applied to investigate the molecular architecture of biomolecular assemblies. High-resolution techniques, such as X-ray crystallography, may solve the atomic structure of the system, but are typically constrained to biomolecules of reduced flexibility and dimensions. In particular, X-ray crystallography requires the sample to form a three dimensional (3D) crystal lattice which is technically di‑cult, if not impossible, to obtain, especially for large, dynamic systems. Often these techniques solve the structure of the different constituent components within the assembly, but encounter difficulties when investigating the entire system. On the other hand, imaging techniques, such as cryo-electron microscopy (cryo-EM), are able to depict large systems in near-native environment, without requiring the formation of crystals. The structures solved by cryo-EM cover a wide range of resolutions, from very low level of detail where only the overall shape of the system is visible, to high-resolution that approach, but not yet reach, atomic level of detail. In this dissertation, several modeling methods are introduced to either integrate cryo-EM datasets with structural data from X-ray crystallography, or to directly interpret the cryo-EM reconstruction. Such computational techniques were developed with the goal of creating an atomic model for the cryo-EM data. The low-resolution reconstructions lack the level of detail to permit a direct atomic interpretation, i.e. one cannot reliably locate the atoms or amino-acid residues within the structure obtained by cryo-EM. Thereby one needs to consider additional information, for example, structural data from other sources such as X-ray crystallography, in order to enable such a high-resolution interpretation. Modeling techniques are thus developed to integrate the structural data from the different biophysical sources, examples including the work described in the manuscript I and II of this dissertation. At intermediate and high-resolution, cryo-EM reconstructions depict consistent 3D folds such as tubular features which in general correspond to alpha-helices. Such features can be annotated and later on used to build the atomic model of the system, see manuscript III as alternative. Three manuscripts are presented as part of the PhD dissertation, each introducing a computational technique that facilitates the interpretation of cryo-EM reconstructions. The first manuscript is an application paper that describes a heuristics to generate the atomic model for the protein envelope of the Rift Valley fever virus. The second manuscript introduces the evolutionary tabu search strategies to enable the integration of multiple component atomic structures with the cryo-EM map of their assembly. Finally, the third manuscript develops further the latter technique and apply it to annotate consistent 3D patterns in intermediate-resolution cryo-EM reconstructions. The first manuscript, titled An assembly model for Rift Valley fever virus, was submitted for publication in the Journal of Molecular Biology. The cryo-EM structure of the Rift Valley fever virus was previously solved at 27Å-resolution by Dr. Freiberg and collaborators. Such reconstruction shows the overall shape of the virus envelope, yet the reduced level of detail prevents the direct atomic interpretation. High-resolution structures are not yet available for the entire virus nor for the two different component glycoproteins that form its envelope. However, homology models may be generated for these glycoproteins based on similar structures that are available at atomic resolutions. The manuscript presents the steps required to identify an atomic model of the entire virus envelope, based on the low-resolution cryo-EM map of the envelope and the homology models of the two glycoproteins. Starting with the results of the exhaustive search to place the two glycoproteins, the model is built iterative by running multiple multi-body refinements to hierarchically generate models for the different regions of the envelope. The generated atomic model is supported by prior knowledge regarding virus biology and contains valuable information about the molecular architecture of the system. It provides the basis for further investigations seeking to reveal different processes in which the virus is involved such as assembly or fusion. The second manuscript was recently published in the of Journal of Structural Biology (doi:10.1016/j.jsb.2009.12.028) under the title Evolutionary tabu search strategies for the simultaneous registration of multiple atomic structures in cryo-EM reconstructions. This manuscript introduces the evolutionary tabu search strategies applied to enable a multi-body registration. This technique is a hybrid approach that combines a genetic algorithm with a tabu search strategy to promote the proper exploration of the high-dimensional search space. Similar to the Rift Valley fever virus, it is common that the structure of a large multi-component assembly is available at low-resolution from cryo-EM, while high-resolution structures are solved for the different components but lack for the entire system. Evolutionary tabu search strategies enable the building of an atomic model for the entire system by considering simultaneously the different components. Such registration indirectly introduces spatial constrains as all components need to be placed within the assembly, enabling the proper docked in the low-resolution map of the entire assembly. Along with the method description, the manuscript covers the validation, presenting the benefit of the technique in both synthetic and experimental test cases. Such approach successfully docked multiple components up to resolutions of 40Å. The third manuscript is entitled Evolutionary Bidirectional Expansion for the Annotation of Alpha Helices in Electron Cryo-Microscopy Reconstructions and was submitted for publication in the Journal of Structural Biology. The modeling approach described in this manuscript applies the evolutionary tabu search strategies in combination with the bidirectional expansion to annotate secondary structure elements in intermediate resolution cryo-EM reconstructions. In particular, secondary structure elements such as alpha helices show consistent patterns in cryo-EM data, and are visible as rod-like patterns of high density. The evolutionary tabu search strategy is applied to identify the placement of the different alpha helices, while the bidirectional expansion characterizes their length and curvature. The manuscript presents the validation of the approach at resolutions ranging between 6 and 14Å, a level of detail where alpha helices are visible. Up to resolution of 12 Å, the method measures sensitivities between 70-100% as estimated in experimental test cases, i.e. 70-100% of the alpha-helices were correctly predicted in an automatic manner in the experimental data. The three manuscripts presented in this PhD dissertation cover different computation methods for the integration and interpretation of cryo-EM reconstructions. The methods were developed in the molecular modeling software Sculptor (http://sculptor.biomachina.org) and are available for the scientific community interested in the multi-resolution modeling of cryo-EM data. The work spans a wide range of resolution covering multi-body refinement and registration at low-resolution along with annotation of consistent patterns at high-resolution. Such methods are essential for the modeling of cryo-EM data, and may be applied in other fields where similar spatial problems are encountered, such as medical imaging.

FUNCTIONS OF DEADENYLATION FACTORS IN MRNA DECAY AND MRNA PROCESSING BODY FORMATION

Most newly synthesized messenger RNAs possess a 5’ cap and a 3’ poly(A) tail. The process of poly(A) tail shortening, also termed deadenylation, is important for post-transcriptional gene regulation, because deadenylation not only leads to mRNA translational inhibition but also is the first step of major mRNA degradation. Translationally inhibited mRNAs can be stored and/or degraded in dynamic cytoplasmic foci termed mRNA processing bodies, or P bodies, which are conserved in eukaryotes. To shed new light on the mechanisms of P body formation and P body functions, I focused on the link between deadenylation factors and P bodies. I found that the two major deadenylation complexes, Pan3-Pan2 and Ccr4-Caf1, can both be enriched in P bodies. The deadenylase activity of the Ccr4-Caf1 complex is prerequisite for P body formation. Pan3, but not the deadenylase Pan2, is essential for P body formation. While the C-terminal domain of Pan3 is important for interaction with Pan2, Pan3 N-terminal domain is important for Pan3 to form cytoplasmic foci colocalizing with P bodies and to promote mRNA decay. Interestingly, Pan3 N-terminal domain may be phosphorylated to regulate Pan3 localization and functions. Aside from the functions of the two deadenylation complexes in P bodies, I also studied all reported human P body proteins as a whole using bioinformatics. This effort not only has generated a comprehensive picture of the functions of and interactions among human P body proteins, but also has predicted proteins that may regulate P body formation and/or functions. In summary, my study has established a direct link between mRNA deadenylation and P body formation and has also led to new hypotheses to guide future research on how P body dynamics are controlled.

Ago-TNRC6 triggers microRNA-mediated decay by promoting two deadenylation steps.

MicroRNAs (miRNAs) silence the expression of their mRNA targets mainly by promoting mRNA decay. The mechanism, kinetics and participating enzymes for miRNA-mediated decay in mammalian cells remain largely unclear. Combining the approaches of transcriptional pulsing, RNA tethering, overexpression of dominant-negative mutants, and siRNA-mediated gene knockdown, we show that let-7 miRNA-induced silencing complexes (miRISCs), which contain the proteins Argonaute (Ago) and TNRC6 (also known as GW182), trigger very rapid mRNA decay by inducing accelerated biphasic deadenylation mediated by Pan2-Pan3 and Ccr4-Caf1 deadenylase complexes followed by Dcp1-Dcp2 complex-directed decapping in mammalian cells. When tethered to mRNAs, all four human Ago proteins and TNRC6C are each able to recapitulate the two deadenylation steps. Two conserved human Ago2 phenylalanines (Phe470 and Phe505) are critical for recruiting TNRC6 to promote deadenylation. These findings indicate that promotion of biphasic deadenylation to trigger mRNA decay is an intrinsic property of miRISCs.

The roles of UPF3a and UPF3b in nonsense mediated decay

Nonsense-mediated decay (NMD) degrades aberrant transcripts containing premature termination codons (PTCs). The T-cell receptor (TCR) locus undergoes error-prone rearrangements that frequently acquire PTCs. Transcripts harboring PTCs from this locus are downregulated much more than transcripts from non-rearranging genes. Efficient splicing is essential for this robust downregulation. ^ Here I show that TCR NMD is unique in another respect: it is not impaired by RNAi-mediated depletion of the NMD factor UPF3b. This differentiates TCR transcripts from classical NMD (assayed using β-globin or triose phosphate isomerase transcripts), which does depend on UPF3b. Depletion of UPF3a, which encodes a gene related to UPF3b, also had no effect on TCR NMD. Mapping experiments identified TCR sequences that when deleted or mutated caused a switch to UPF3b dependence. Since UPF3b dependence was invariably accompanied by less efficient RNA splicing, this suggests that UPF3b-dependent NMD occurs when transcripts are generated by inefficient splicing. Microarray analysis revealed the existence of many NMD-targeted mRNAs from wild-type genes whose downregulation is impervious to UPF3b depletion. This suggests the existence of an alternative NMD pathway independent of UPF3b that is widely used to downregulate the level of both normal and mutant transcripts. ^ During the course of my studies, I also found that the function of UPF3a is fundamentally distinct from that of UPF3b in several aspects. First, classical NMD failed to be impaired by UPF3a depletion, whereas it was reversed by UPF3b depletion. Second, UPF3a depletion had no effect on NMD elicited by tethered UPF2, whereas UPF3b depletion blocked this response. Thus, UPF3a does not function in classical NMD. Third, UPF3b depletion upregulated the expression of UPF3a, whereas UPF3a depletion had no effect on UPF3b expression. This suggests that a UPF3b-mediated feedback network exists that regulates the UPF3a expression. Lastly, UPF3a depletion but not UPF3b depletion significantly upregulated TCR precursor RNAs. This suggests that UPF3a, not UPF3b, functions in the surveillance of precursor RNAs, which typically contain many PTCs in the introns. Collectively, my data suggests that UPF3a and UPF3b are not functionally redundant, as previously thought, but instead have separable functions. ^