RADAR: a web server for RNA data analysis and research

RADAR is a web server that provides a multitude of functionality for RNA data analysis and research. It can align structure-annotated RNA sequences so that both sequence and structure information are taken into consideration during the alignment process. This server is capable of performing pairwise structure alignment, multiple structure alignment, database search and clustering. In addition, RADAR provides two salient features: (i) constrained alignment of RNA secondary structures, and (ii) prediction of the consensus structure for a set of RNA sequences. RADAR will be able to assist scientists in performing many important RNA mining operations, including the understanding of the functionality of RNA sequences, the detection of RNA structural motifs and the clustering of RNA molecules, among others. The web server together with a software package for download is freely accessible at http://datalab.njit.edu/biodata/rna/RSmatch/server.htm and http://www.ccrnp.ncifcrf.gov/~bshapiro

Predicting Consensus Structures for RNA Alignments Via Pseudo-Energy Minimization

Thermodynamic processes with free energy parameters are often used in algorithms that solve the free energy minimization problem to predict secondary structures of single RNA sequences. While results from these algorithms are promising, an observation is that single sequence-based methods have moderate accuracy and more information is needed to improve on RNA secondary structure prediction, such as covariance scores obtained from multiple sequence alignments. We present in this paper a new approach to predicting the consensus secondary structure of a set of aligned RNA sequences via pseudo-energy minimization. Our tool, called RSpredict, takes into account sequence covariation and employs effective heuristics for accuracy improvement. RSpredict accepts, as input data, a multiple sequence alignment in FASTA or ClustalW format and outputs the consensus secondary structure of the input sequences in both the Vienna style Dot Bracket format and the Connectivity Table format. Our method was compared with some widely used tools including KNetFold, Pfold and RNAalifold. A comprehensive test on different datasets including Rfam sequence alignments and a multiple sequence alignment obtained from our study on the Drosophila X chromosome reveals that RSpredict is competitive with the existing tools on the tested datasets. RSpredict is freely available online as a web server and also as a jar file for download at http://datalab.njit.edu/biology/RSpredict

The Non-Canonical CTD of RNAP-II Is Essential for Productive RNA Synthesis in Trypanosoma brucei

The carboxy-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II (RNAP-II) is essential for gene expression in metazoa and yeast. The canonical CTD is characterized by heptapeptide repeats. Differential phosphorylation of canonical CTD orchestrates transcriptional and co-transcriptional maturation of mRNA and snRNA. Many organisms, including trypanosomes, lack a canonical CTD. In these organisms, the CTD is called a non-canonical CTD or pseudo-CTD (ΨCTD. In the African trypanosome, Trypanosoma brucei, the ΨCTD is ∼285 amino acids long, rich in serines and prolines, and phosphorylated. We report that T. brucei RNAP-II lacking the entire ΨCTD or containing only a 95-amino-acid-long ΨCTD failed to support cell viability. In contrast, RNAP-II with a 186-amino-acid-long ΨCTD maintained cellular growth. RNAP-II with ΨCTD truncations resulted in abortive initiation of transcription. These data establish that non-canonical CTDs play an important role in gene expression

A leucine aminopeptidase is involved in kinetoplast DNA segregation in <i>Trypanosoma brucei</i>

The kinetoplast (k), the uniquely packaged mitochondrial DNA of trypanosomatid protists is formed by a catenated network of minicircles and maxicircles that divide and segregate once each cell cycle. Although many proteins involved in kDNA replication and segregation are now known, several key steps in the replication mechanism remain uncharacterized at the molecular level, one of which is the nabelschnur or umbilicus, a prominent structure which in the mammalian parasite Trypanosoma brucei connects the daughter kDNA networks prior to their segregation. Here we characterize an M17 family leucyl aminopeptidase metalloprotease, termed TbLAP1, which specifically localizes to the kDNA disk and the nabelschur and represents the first described protein found in this structure. We show that TbLAP1 is required for correct segregation of kDNA, with knockdown resulting in delayed cytokinesis and ectopic expression leading to kDNA loss and decreased cell proliferation. We propose that TbLAP1 is required for efficient kDNA division and specifically participates in the separation of daughter kDNA networks

Characterization of deadenylation in trypanosome extracts and its inhibition by poly(A)-binding protein Pab1p

The stability of mRNAs is an important point in the regulation of gene expression in eukaryotes. The mRNA turnover pathways have been identified in yeast and mammals. However, mRNA turnover pathways in trypanosomes have not been widely studied. Deadenylation is the first step in the major mRNA turnover pathways of yeast and mammals. To better understand mRNA degradation processes in these organisms, we have developed an in vitro mRNA turnover system that is functional for deadenylation. In this system, addition of poly(A) homopolymer activates the deadenylation of poly(A) tails. The trypanosomal deadenylase activity is a 3′→5′ exonuclease specific for adenylate residues, generates 5′-AMP as a product, is magnesium dependent, and is inhibited by neomycin B sulfate. These characteristics suggest similarity with other eukaryotic deadenylases. Furthermore, this activity is cap independent, indicating a potential difference between the trypanosomal activity and PARN, but suggesting similarity to Ccr4p/Pop2p activities. Extracts immunodepleted of Pab1p required the addition of poly(A) competition to activate deadenylation. Trypanosomal Pab1p functions as an inhibitor of the activity under in vitro conditions. Pab1p appears to be one of several mRNA stability proteins in trypanosomal extracts