Human telomerase reverse transcriptase binds to a pre-organized hTR in vivo exposing its template.

Telomerase is a specialized reverse transcriptase that is responsible for telomere length maintenance. As in other organisms, the minimal components required for an active human telomerase are the template-providing telomerase RNA (hTR) and the enzymatic entity telomerase reverse transcriptase (hTERT). Here, we explored the structure of hTR and the hTERT-induced conformational changes within hTR in living cells.

Chemical probing of RNA in living cells.

RNAs need to adopt a specific architecture to exert their task in cells. While significant progress has been made in describing RNA folding landscapes in vitro, understanding intracellular RNA structure formation is still in its infancy. This is in part due to the complex nature of the cellular environment but also to the limited availability of suitable methodologies.

Mapping RNA structure in vitro using nucleobase-specific probes.

RNAs have to adopt specific three-dimensional structures to fulfill their biological functions. Therefore exploring RNA structure is of interest to understand RNA-dependent processes. Chemical probing in vitro is a very powerful tool to investigate RNA molecules under a variety of conditions.

Insights into diseases of human telomerase from dynamical modeling.

Mutations in the telomerase complex disrupt either nucleic acid binding or catalysis, and are the cause of numerous human diseases. Despite its importance, the structure of the human telomerase complex has not been observed crystallographically, nor are its dynamics understood in detail. Fragments of this complex from Tetrahymena thermophila and Tribolium castaneum have been crystallized.

Mss116p: a DEAD-box protein facilitates RNA folding.

RNA folding is an essential aspect underlying RNA-mediated cellular processes. Many RNAs, including large, multi-domain ribozymes, are capable of folding to the native, functional state without assistance of a protein cofactor in vitro. In the cell, trans-acting factors, such as proteins, are however known to modulate the structure and thus the fate of an RNA.

RNA-Puzzles: a CASP-like evaluation of RNA three-dimensional structure prediction.

We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools.

A structural determinant required for RNA editing.

RNA editing by adenosine deaminases acting on RNAs (ADARs) can be both specific and non-specific, depending on the substrate. Specific editing of particular adenosines may depend on the overall sequence and structural context. However, the detailed mechanisms underlying these preferences are not fully understood.

DEAD-box protein facilitated RNA folding in vivo.

In yeast mitochondria the DEAD-box helicase Mss116p is essential for respiratory growth by acting as group I and group II intron splicing factor. Here we provide the first structure-based insights into how Mss116p assists RNA folding in vivo. Employing an in vivo chemical probing technique, we mapped the structure of the ai5γ group II intron in different genetic backgrounds to characterize its intracellular fold.

RNA folding in living cells.

RNA folding is the most essential process underlying RNA function. While significant progress has been made in understanding the forces driving RNA folding in vitro, exploring the rules governing intracellular RNA structure formation is still in its infancy. The cellular environment hosts a great diversity of factors that potentially influence RNA folding in vivo.

Probing RNA structure within living cells.

RNA folding is the most fundamental process underlying RNA function. RNA structure and associated folding paradigms have been intensively studied in vitro. However, in vivo RNA structure formation has only been explored to a limited extent.

Dissecting RNA folding by nucleotide analog interference mapping (NAIM).

Nucleotide analog interference mapping (NAIM) is a powerful chemogenetic approach that allows RNA structure and function to be characterized at the atomic level. Random modifications of base or backbone moieties are incorporated into the RNA transcript as nucleotide analog phosphorothioates. The resulting RNA pool is then subjected to a stringent selection step, in which the RNA has to accomplish a specific task, for example, folding.

RNA chaperones, RNA annealers and RNA helicases.

RNA molecules face difficulties when folding into their native structures. In the cell, proteins can assist RNAs in reaching their functionally active states by binding and stabilizing a specific structure or, in a quite opposite way, by interacting in a non-specific manner. These proteins can either facilitate RNA-RNA interactions in a reaction termed RNA annealing, or they can resolve non-functional inhibitory structures.

A kinetic intermediate that regulates proper folding of a group II intron RNA.

The D135 group II intron ribozyme follows a unique folding pathway that is direct and appears to be devoid of kinetic traps. During the earliest stages of folding, D135 collapses slowly to a compact intermediate, and all subsequent assembly events are rapid. Collapse of intron domain 1 (D1) has been shown to limit the rate constant for D135 folding, although the specific substructure of the D1 kinetic intermediate has not yet been identified.

Folding of group II introns: a model system for large, multidomain RNAs?

Group II introns are among the largest ribozymes in nature. They have a highly complex tertiary architecture that enables them to catalyze numerous processes, including self-splicing and transposition reactions that have probably contributed to the evolution of eukaryotic genomes. Biophysical analyses show that, despite their large size, these RNAs can fold to their native state through direct pathways that are populated by structurally defined intermediates.

Group II intron folding under near-physiological conditions: collapsing to the near-native state.

The folding of group II intron ribozymes has been studied extensively under optimal conditions for self-splicing in vitro (42 degrees C and high magnesium ion concentrations). In these cases, the ribozymes fold directly to the native state by an apparent two-state mechanism involving the formation of an obligate intermediate within intron domain 1. We have now characterized the folding pathway under near-physiological conditions.

A folding control element for tertiary collapse of a group II intron ribozyme.

Ribozymes derived from the group II intron ai5gamma collapse to a compact intermediate, folding to the native state through a slow, direct pathway that is unperturbed by kinetic traps. Molecular collapse of ribozyme D135 requires high magnesium concentrations and is thought to involve a structural element in domain 1 (D1). We used nucleotide analog interference mapping, in combination with nondenaturing gel electrophoresis, to identify RNA substructures and functional groups that are essential for D135 tertiary collapse.

An obligate intermediate along the slow folding pathway of a group II intron ribozyme.

Most RNA molecules collapse rapidly and reach the native state through a pathway that contains numerous traps and unproductive intermediates. The D135 group II intron ribozyme is unusual in that it can fold slowly and directly to the native state, despite its large size and structural complexity. Here we use hydroxyl radical footprinting and native gel analysis to monitor the timescale of tertiary structure collapse and to detect the presence of obligate intermediates along the folding pathway of D135.

Influence of RNA structural stability on the RNA chaperone activity of the Escherichia coli protein StpA.

Proteins with RNA chaperone activity are able to promote folding of RNA molecules by loosening their structure. This RNA unfolding activity is beneficial when resolving misfolded RNA conformations, but could be detrimental to RNAs with low thermodynamic stability. In order to test this idea, we constructed various RNAs with different structural stabilities derived from the thymidylate synthase (td) group I intron and measured the effect of StpA, an Escherichia coli protein with RNA chaperone activity, on their splicing activity in vivo and in vitro.

Monitoring intermediate folding states of the td group I intron in vivo.

Group I introns consist of two major structural domains, the P4-P6 and P3-P9 domains, which assemble through interactions with peripheral extensions to fold into an active ribozyme. To assess group I intron folding in vivo, we probed the structure of td wild-type and mutant introns using dimethyl sulfate. The results suggest that the majority of the intron population is in the native state in accordance with the current structural model, which was refined to include two novel tertiary contacts.

RNA chaperone StpA loosens interactions of the tertiary structure in the td group I intron in vivo.

Efficient splicing of the td group I intron in vivo is dependent on the ribosome. In the absence of translation, the pre-mRNA is trapped in nonnative-splicing-incompetent conformations. Alternatively, folding of the pre-mRNA can be promoted by the RNA chaperone StpA or by the group I intron-specific splicing factor Cyt-18.

RNA folding in vivo.

RNA folding in vivo is influenced by the cellular environment, the vectorial nature of transcription and translation, trans-acting factors and ion homeostasis. Specific RNA-binding proteins promote RNA folding by stabilizing the native structure or by guiding folding. In contrast, RNA chaperones, which are believed to interact nonspecifically with RNA, were proposed to resolve misfolded RNA structures and to promote intermolecular RNA-RNA annealing.