Patterns of selective constraints in noncoding DNA of rice

<p>Abstract</p> <p>Background</p> <p>Several studies have investigated the relationships between selective constraints in introns and their length, GC content and location within genes. To date, however, no such investigation has been done in plants. Studies of selective constraints in noncoding DNA have generally involved interspecific comparisons, under the assumption of the same selective pressures acting in each lineage. Such comparisons are limited to cases in which the noncoding sequences are not too strongly diverged so that reliable sequence alignments can be obtained. Here, we investigate selective constraints in a recent segmental duplication that includes 605 paralogous intron pairs that occurred about 7 million years ago in rice (<it>O. sativa</it>).</p> <p>Results</p> <p>Our principal findings are: (1) intronic divergence is negatively correlated with intron length, a pattern that has previously been described in <it>Drosophila </it>and mammals; (2) there is a signature of strong purifying selection at splice control sites; (3) first introns are significantly longer and have a higher GC content than other introns; (4) the divergences of first and non-first introns are not significantly different from one another, a pattern that differs from <it>Drosophila </it>and mammals; and (5) short introns are more diverged than four-fold degenerate sites suggesting that selection reduces divergence at four-fold sites.</p> <p>Conclusion</p> <p>Our observation of stronger selective constraints in long introns suggests that functional elements subject to purifying selection may be concentrated within long introns. Our results are consistent with the presence of strong purifying selection at splicing control sites. Selective constraints are not significantly stronger in first introns of rice, as they are in other species.</p

The A-rich RNA sequences of HIV-1 pol are important for the synthesis of viral cDNA

The bias of A-rich codons in HIV-1 pol is thought to be a record of hypermutations in viral genomes that lack biological functions. Bioinformatic analysis predicted that A-rich sequences are generally associated with minimal local RNA structures. Using codon modifications to reduce the amount of A-rich sequences within HIV-1 genomes, we have reduced the flexibility of RNA sequences in pol to analyze the functional significance of these A-rich ‘structurally poor’ RNA elements in HIV-1 pol. Our data showed that codon modification of HIV-1 sequences led to a suppression of virus infectivity by 5–100-fold, and this defect does not correlate with, viral entry, viral protein expression levels, viral protein profiles or virion packaging of genomic RNA. Codon modification of HIV-1 pol correlated with an enhanced dimer stability of the viral RNA genome, which was associated with a reduction of viral cDNA synthesis both during HIV-1 infection and in a cell free reverse transcription assay. Our data provided direct evidence that the HIV-1 A-rich pol sequence is not merely an evolutionary artifact of enzyme-induced hypermutations, and that HIV-1 has adapted to rely on A-rich RNA sequences to support the synthesis of viral cDNA during reverse transcription, highlighting the utility of using ‘structurally poor’ RNA domains in regulating biological process

MOLECULAR DYNAMICS STUDIES OF NUCLEIC ACIDS AND RIBONUCLEOPROTEIN COMPLEXES

Molecular simulations of protein-nucleic acid complexes, as well as the HIV-1 Trans Activation Response Element (TAR) RNA molecule, were conducted. First, three different molecular dynamics techniques were studied on the molecule HIV-1 TAR RNA. The three techniques studied were classical molecular dynamics, steered molecular dynamics (SMD), and metadynamics. The classic molecular dynamics simulations were used to equilibrate the HIV-1 TAR RNA system, as well as every other system studied in this thesis. The SMD technique was used in order to observe the breaking force of the nucleotide interactions within TAR. This breaking force averaged to about 100pN. The metadynamics technique was used in order to accelerate the folding of HIV-1 TAR RNA from an unfolded state to its native state. With the use of root mean square deviation (RMSD) and radius of gyration (RGYR) as collective variables (CVs) we were not able to successfully fold HIV-1 TAR RNA xiv from an unfolded state to it’s native state, however, we did obtain four unique conformations of TAR that were within 1kcal/mol of the native state in free energy. Next, the classification of interaction strength between nine diverse nucleic acidprotein complexes was studied using the SMD technique. The nine chosen complexes vary in size (800-6000 atoms) as well as in the type of RNA binding protein (RBP) bound to RNA. In these simulations the RNA molecule in each system is partially fixed and the protein atoms in the binding interface are pulled at a constant velocity. Force data is obtained for each of the nine systems and the maximum force required to separate the molecules is compared using two different variables, percent composition of charged amino acid residues in the binding interface (percent composition) and buried surface area (BSA). We also look at the van der Waals and electrostatic interactions of each system over their respective trajectories. It was found that an increase in BSA often resulted in a higher value of the maximum force. The percent composition did not correlate well with the maximum force, however it is shown that the arginine rich motif (1ETG) system surprisingly had a relatively high maximum force value for such a small BSA and system size. Lastly, the binding affinity of an arginine residue bound to RNA and an adenine monophosphate (AMP) molecule bound to RNA is determined using the well-tempered metadynamics technique. Binding affinity is an important aspect to drug targeting. An effective characterization of a molecules binding affinity is the free energy of binding. Finding a way to calculate this value using molecular dynamics simulations could save much time in the drug development process. We apply well-tempered metadynamics to two small molecule systems that resemble drug-like molecular systems in order to xv determine the binding free energy of these systems. The aim here was to first test the technique on these two example systems such that the same process could be repeated for any system involving the binding of drug molecules to proteins or nucleic acids. Using welltempered metadynamics with a center-of-mass distance CV we were able to successfully determine the binding free energy of the two model systems

Evolutionary patterns of non-coding RNAs

A plethora of new functions of non-coding RNAs have been discovered in past few years. In fact, RNA is emerging as the central player in cellular regulation, taking on active roles in multiple regulatory layers from transcription, RNA maturation, and RNA modification to translational regulation. Nevertheless, very little is known about the evolution of this \Modern RNA World' and its components. In this contribution we attempt to provide at least a cursory overview of the diversity of non-coding RNAs and functional RNA motifs in non-translated regions of regular messenger RNAs (mRNAs) with an emphasis on evolutionary questions. This survey is complemented by an in-depth analysis of examples from different classes of RNAs focusing mostly on their evolution in the vertebrate lineage. We present a survey of Y RNA genes in vertebrates, studies of the molecular evolution of the U7 snRNA, the snoRNAs E1/U17, E2, and E3, the Y RNA family, the let-7 microRNA family, and the mRNA-like evf-1 gene. We furthermore discuss the statistical distribution of microRNAs in metazoans, which suggests an explosive increase in the microRNA repertoire in vertebrates. The analysis of the transcription of non-coding RNAs (ncRNAs) suggests that small RNAs in general are genetically mobile in the sense that their association with a hostgene (e.g. when transcribed from introns of a mRNA) can change on evolutionary time scales. The let-7 family demonstrates, that even the mode of transcription (as intron or as exon) can change among paralogous ncRNA

Regulation of translation during cancer progression

Laminin B1 (LamB1) ist ein Bestandteil der extrazellulären Matrix und auf entscheidende Weise in der Regulation der Tumorzellmigration und –invasion während der Karzinogenese involviert. Ein essentieller Aspekt der Tumorprogression ist die Umwandlung epithelialer Tumorzellen zu einem mesenchymalen Phänotyp (EMT), der mit metastatischen Eigenschaften korreliert. Ein Expressionsprofil polysom-assozierter mRNA maligner Hepatozyten zeigte die Aktivierung der Translation von LamB1 in EMT-transformierten Zellen. Es stellte sich heraus, das die erhöhte Translation von LamB1 in metastasierenden Hepatozyten von einer internen Ribosomen-Bindungsstelle (IRES) reguliert wird, welche in der 5‘-untranslatierten Region (UTR) des LamB1 Transkripts lokalisiert ist. Zwei unabhängige bicistronische Reportersysteme wurden für die Bestimmung der IRES-Aktivität von LamB1 verwendet. Zur Verifikation der IRES Aktivität wurde die Präsenz kryptischer Promotoren und Spleißstellen im 5’-UTR von LamB1 ausgeschlossen. Die minimale cis-agierende IRES Sequenz, welche für die „Cap“-unabhängige Translation notwendig ist, wurde mit einer Länge von 293 Nukleotiden direkt oberhalb des Startcodons lokalisiert. Mithilfe einer RNA Affinitätschromatography wurde der IRES trans-aktivierende Faktor (ITAF) La als Bindungspartner identifiziert, welcher mit der LamB1 5’-UTR in vitro interagiert. Diese Interaktion wurde durch eine RNA-Immunopräzipitation in vivo verifiziert. Weiters konnte die Bindung einer erhöhten Menge von La Protein an das minimale LamB1 IRES Motiv nachgewiesen werden. In Übereinstimmung mit dieser Beobachtung konnten erhöhte zytoplasmatische Mengen von La in EMT-transformierten Zellen detektiert werden. Überdies führte die Gegenwart von La zu einer gesteigerten IRES-meditierten Translation von LamB1 in vitro. Platelet derived growth factor (PDGF) wurde als auslösender Signaltransduktionsweg für die zytoplasmatische Translokation von La identifiziert. Zusammenfassend konnte damit nachgewiesen werden, dass der PDGF Signalweg für die zytoplasmatische Akkumulation von La und damit der Interaktion mit dem LamB1 IRES verantwortlich ist, wodurch die cap-unabhängige Translation von LamB1 in malignen Hepatozyten nach EMT aktiviert wird.Laminin B1 (LamB1) is a main component of the extracellular matrix and is involved in the regulation of tumor cell migration and invasion of during carcinogenesis. Metastasis of carcinoma cells is crucially linked to the process of epithelial to mesenchymal transition (EMT), which allows tumor cells to acquire a more motile phenotype and to dissociate from the epithelial cell cluster of the tumor. Expression profiling of polysome-associated mRNA revealed LamB1 to be translationally upregulated upon EMT of malignant hepatocytes. The enhanced translation of LamB1 in metastatic hepatocytes proved to be regulated by an internal ribosome entry site (IRES) located within the 5’-untranslated region (UTR) of the LamB1 transcript. IRES activity was detected by employing two independent reporter systems and verified by stringent assays for the presence of cryptic promoter or splice sites. The minimal cis-acting IRES sequence of 293 nucleotides that is required for cap-independent translation was localized directly upstream of the start codon. Notably, the IRES trans-acting factor (ITAF) La was identified by RNA affinity purification as regulatory factor that interacts with LamB1 5’-UTR. This interaction was verified by RNA-immunoprecipitation in vivo, which revealed enhanced binding of La to the minimal IRES motif of LamB1 after EMT. Consistently, cytoplasmic levels of La were elevated in EMT-transformed cells and correlated with increased LamB1 protein expression. Furthermore, IRES-driven translation of LamB1 was elevated in the presence of La in vitro. Importantly, the EMT-induced cytoplasmic translocation of La was found to be triggered by platelet derived growth factor (PDGF) that is downstream of transforming growth factor (TGF)-β signaling. Together, these data demonstrate that La interacts with the LamB1 IRES in the cytoplasm, resulting in enhanced cap-independent translation of LamB1 in malignant hepatocytes that have undergone EMT