HOTAIR lifts noncoding RNAs to new levels

It is not clear to what extent noncoding RNAs regulate the homeobox (HOX) genes that encode key regulators of development in the embryo. In this issue, Rinn et al. (2007) characterize noncoding RNAs that regulate HOX genes and discover one, HOTAIR, that unexpectedly regulates a HOX gene cluster on a different chromosome than the HOX cluster that encodes it.     

Non-micro-short RNAs: the new kids on the block

The advent of ultra–high-throughput sequencing has led to the discovery of a large group of small, noncoding RNAs that are not microRNAs. The functional relevance of microRNAs has been well established over the last decade. In this Perspective, we focus on the non-micro-short RNAs that comprise a variety of functional classes and range from 16–40 nucleotides in size. We will highlight how some of these non-micro-short RNAs were discovered, as well as their biogenesis, potential mechanisms of action, and role in diverse biological processes, development, and disease. Finally, we will describe what must be done to further our understanding of these enigmatic molecules.     

Regulatory non-coding RNAs: a new frontier in regulation of plant biology

Functional & Integrative Genomics - Beyond the most crucial roles of RNA molecules as a messenger, ribosomal, and transfer RNAs, the regulatory role of many non-coding RNAs (ncRNAs) in plant...     

A new distance for high level RNA secondary structure comparison

We describe an algorithm for comparing two RNA secondary structures coded in the form of trees that introduces two new operations, called node fusion and edge fusion, besides the tree edit operations of deletion, insertion, and relabeling classically used in the literature. This allows us to address some serious limitations of the more traditional tree edit operations when the trees represent RNAs and what is searched for is a common structural core of two RNAs. Although the algorithm complexity has an exponential term, this term depends only on the number of successive fusions that may be applied to a same node, not on the total number of fusions. The algorithm remains therefore efficient in practice and is used for illustrative purposes on ribosomal as well as on other types of RNAs.     

Characterization of three new snRNAs from Saccharomyces cerevisiae: snR34, snR35 and snR36.

Genes for three novel snRNAs of Saccharomyces cerevisiae have been isolated, sequenced and tested for essentiality. The RNAs encoded by these genes are designated snR34, snR35 and snR36 respectively and contain 203, 204 and 182 nucleotides. Each RNA is derived from a single copy gene and all three RNAs are believed to be nucleolar, i.e. snoRNAs, based on extraction properties and association with fibrillarin. SnR34 and snR35 contain a trimethylguanosine cap, but this feature is absent from snR36. The novel RNAs lack elements conserved among several other snoRNAs, including box C, box D and long sequence complementarities with rRNA. Genetic disruption analyses showed each of the RNAs to be dispensable and a haploid strain lacking all three RNAs and a previously characterized fourth snoRNA (snR33) is also viable. No differences in the levels of precursors or mature rRNAs were apparent in the four gene knock-out strain. Possible roles for the new RNAs in ribosome biogenesis are discussed.     

Identifying expression of new small RNAs by microarrays

Although a large number of small RNAs (sRNAs) have been discovered, it is very likely that the screens conducted so far have not reached saturation. Recently, many methods for predicting and identifying new sRNAs have been developed. However, it remains unclear what the total number of sRNAs within a genome is and how many types of sRNAs exist in plants and animals. In this article, combined methods of dynamic programming prediction, enrichment of sRNAs, and microarray analysis are developed to screen and evaluate a new class of sRNAs from introns of human, protein-encoding genes. The methods used by our laboratories to design capture probes and label enriched small RNAs are thoroughly described here. The microarray results show that our modified technologies are useful to enhance sensitivity and specificity of arrays, identify expression patterns within different cells, and discover differential expression of sRNAs during the differentiation process of bone marrow stem cells. Accordingly, the combination of computational prediction and microarray analysis may be a feasible and practical approach for profiling studies of both known and predicted small RNAs.     

Pseudoknots: a new motif in the RNA game.

In the last few years a novel RNA folding principle called pseudoknotting has emerged. Originally discovered in noncoding regions of plant viral RNAs, pseudoknots now appear to be a widespread structural motif in a number of functionally different RNAs. These structural elements are part of tRNA-like structures and are involved in folding catalytic sites of ribozymes. They increase the efficiency of ribosomal frameshifting or can serve as specific binding sites for regulatory proteins.     

Heavy metal-regulated new microRNAs from rice

MicroRNAs (miRNAs) are a novel class of short, endogenous non-coding small RNAs that have the ability to base pair with their target mRNAs to repress their translation or induce their degradation in both plants and animals. To identify heavy metal stress-regulated novel miRNAs, we constructed a library of small RNAs from rice seedlings that were exposed to toxic levels of cadmium (Cd²⁺). Sequencing of the library and subsequent analysis revealed 19 new miRNAs representing six families. These cloned new rice miRNAs have sequence conservation neither in Arabidopsis nor in any other species. Most of the new rice miRNAs were up- or down-regulated in response to the metal exposure. On the base of sequence complementarity, a total of 34 miRNA targets were predicted, of which 23 targets are functionally annotated and the other 11 records belong to unknown proteins. Some predicted targets of miRNAs are associated with the regulation of the response to heavy metal-induced stresses. In addition to the new miRNAs, we detected nine previously reported miRNAs and 56 other novel endogenous small RNAs in rice. These findings suggest that the number of new miRNAs in rice is unsaturated and some of them may play critical roles in plant responses to environmental stresses.     

Consensus folding of aligned sequences as a new measure for the detection of functional RNAs by comparative genomics

Facing the ever-growing list of newly discovered classes of functional RNAs, it can be expected that further types of functional RNAs are still hidden in recently completed genomes. The computational identification of such RNA genes is, therefore, of major importance. While most known functional RNAs have characteristic secondary structures, their free energies are generally not statistically significant enough to distinguish RNA genes from the genomic background. Additional information is required. Considering the wide availability of new genomic data of closely related species, comparative studies seem to be the most promising approach. Here, we show that prediction of consensus structures of aligned sequences can be a significant measure to detect functional RNAs. We report a new method to test multiple sequence alignments for the existence of an unusually structured and conserved fold. We show for alignments of six types of well-known functional RNA that an energy score consisting of free energy and a covariation term significantly improves sensitivity compared to single sequence predictions. We further test our method on a number of non-coding RNAs from Caenorhabditis elegans/Caenorhabditis briggsae and seven Saccharomyces species. Most RNAs can be detected with high significance. We provide a Perl implementation that can be used readily to score single alignments and discuss how the methods described here can be extended to allow for efficient genome-wide screens.     

A new sensitive radial diffusion method for microdetermination of alpha-amylase

A rapid, convenient, and efficient hybridization method for the determination of virus-specific RNAs in Py virus-infected cells is described. The method involves carrying out the hybridization of viral RNAs present in the RNA isolated from infected cells directly on nitrocellulose filters carrying denatured, immobilized Py-DNA (15 μl RNA solution/25-mm² filter). Under optimal conditions quantitative hybridization of viral RNA sequences is obtained within 24 h. The efficiency of hybridization is increased significantly when RNA fragmented by alkali under carefully controlled conditions is used.     

U3, U8 and U13 comprise a new class of mammalian snRNPs localized in the cell nucleolus.

Using anti-(U3)RNP autoantibodies, we have isolated and characterized two additional small nucleolar RNAs from HeLa cells, which are less abundant than U3 RNA. Both RNAs possess a trimethylguanosine cap as judged by precipitation with anti-TMG antibody, but are not precipitated by either anti-Sm or anti-La antibodies. In addition, both RNAs are not precipitable by anti-Th serum, which recognizes another nucleolar RNP autoantigen. Sequence analysis revealed that one of these RNAs, 136 nucleotides long, is the human U8 homolog; while the other, 105 nucleotides long, represents a novel species which we designate U13. Both RNAs share with U3 two conserved sequences (boxes C and D). The role of one or both of these boxes in binding the common 34 kd antigenic protein, otherwise known as fibrillarin, is discussed. Fractionation of HeLa cells revealed that U8 and U13, like U3, reside in the nucleolus. In glycerol gradients both RNAs cosediment with larger structures possibly representing ribosomal precursors. We propose that U3, U8 and U13 comprise a new subset of mammalian snRNPs whose roles in ribosome biogenesis are discussed.     

Three new small nucleolar RNAs that are psoralen cross-linked in vivo to unique regions of pre-rRNA.

We have recently described three novel human small nucleolar RNA species with unique nucleotide sequences, which were named E1, E2, and E3. The present article describes specific psoralen photocross-linking in whole HeLa cells of E1, E2, and E3 RNAs to nucleolar pre-rRNA. These small RNAs were cross-linked to different sections of pre-rRNA. E1 RNA was cross-linked to two segments of nucleolar pre-rRNA; one was within residues 697 to 1163 of the 5' external transcribed spacer, and the other one was between nucleotides 664 and 1021 of the 18S rRNA sequence. E2 RNA was cross-linked to a region within residues 3282 to 3667 of the 28S rRNA sequence. E3 RNA was cross-linked to a sequence between positions 1021 and 1639 of the 18S rRNA sequence. Primer extension analysis located psoralen adducts in E1, E2, and E3 RNAs that were enriched in high-molecular-weight fractions of nucleolar RNA. Some of these psoralen adducts might be cross-links of E1, E2, and E3 RNAs to large nucleolar RNA. Antisense oligodeoxynucleotide-targeted RNase H digestion of nucleolar extracts revealed accessible segments in these three small RNAs. The accessible regions were within nucleotide positions 106 to 130 of E1 RNA, positions 24 to 48 and 42 to 66 of E2 RNA, and positions 7 to 16 and about 116 to 122 of E3 RNA. Some of the molecules of these small nucleolar RNAs sedimented as if associated with larger structures when both nondenatured RNA and a nucleolar extract were analyzed.     

Mining small RNA sequencing data: a new approach to identify small nucleolar RNAs in Arabidopsis

Small nucleolar RNAs (snoRNAs) are noncoding RNAs that direct 2′-O-methylation or pseudouridylation on ribosomal RNAs or spliceosomal small nuclear RNAs. These modifications are needed to modulate the activity of ribosomes and spliceosomes. A comprehensive repertoire of snoRNAs is needed to expand the knowledge of these modifications. The sequences corresponding to snoRNAs in 18–26-nt small RNA sequencing data have been rarely explored and remain as a hidden treasure for snoRNA annotation. Here, we showed the enrichment of small RNAs at Arabidopsis snoRNA termini and developed a computational approach to identify snoRNAs on the basis of this characteristic. The approach successfully uncovered the full-length sequences of 144 known Arabidopsis snoRNA genes, including some snoRNAs with improved 5′- or 3′-end annotation. In addition, we identified 27 and 17 candidates for novel box C/D and box H/ACA snoRNAs, respectively. Northern blot analysis and sequencing data from parallel analysis of RNA ends confirmed the expression and the termini of the newly predicted snoRNAs. Our study especially expanded on the current knowledge of box H/ACA snoRNAs and snoRNA species targeting snRNAs. In this study, we demonstrated that the use of small RNA sequencing data can increase the complexity and the accuracy of snoRNA annotation.     

Argonautes confront new small RNAs

Argonaute is at the heart of all effector complexes in RNA interference. In the classical RNAi pathway Argonaute functions as the Slicer enzyme that cleaves an mRNA target directed by a complementary siRNA. Two recently described Argonaute protein subfamilies mediate distinct functions in RNAi. The Piwi subfamily functions in the germline through a novel class of small RNAs that are longer than Argonaute-specific siRNAs and miRNAs. Piwi-interacting RNAs (piRNAs) carry a 2'-O-methylation on their 3' end and appear to be synthesized by a Piwi Slicer dependent mechanism. Piwi/piRNA complexes in mammals and flies are directly linked to the control of transposable elements during germline development. Amplified RNAi in C. elegans is mediated by secondary siRNAs selectively bound to secondary Argonautes (SAGOs) that belong to a worm-specific Argonaute subfamily (WAGO). Secondary siRNAs are 5' triphosphorylated that may allow specific loading into SAGO complexes that are rate limiting for RNAi in C. elegans. Interestingly, SAGOs lack conserved Slicer amino acid residues and probably act in a Slicer-independent fashion.     

A new series of RNAs associated with the genome of spleen focus forming virus (SFFV) and poly(A)-containing RNA from SFFV-infected cells.

A series of low molecular weight RNAs (4.5 to 5.5S) as well as other 4 to 7S RNAs were dissociated from genomic RNA of spleen focus forming virus (SFFV) by heating. On two dimensional polyacrylamide gel electrophoresis, this series of RNAs gave a series of more than thirty spots. RNase T1 fingerprints of these spots were identical except for differences in 3'-terminal oligonucleotides, which were mainly due to different numbers of uridylic acid residues, larger RNA-molecules containing poly(U)sequences at their 3'-termini. This series of RNAs is also associated with poly(A)-containing nuclear and cytoplasmic RNAs from SFFV-infected cells.