ATP-dependent chromatin remodeling shapes the long noncoding RNA landscape.

Long noncoding RNAs (lncRNAs) are pervasively transcribed across eukaryotic genomes, but functions of only a very small subset of them have been demonstrated. This has led to active debate about whether many of them have any biological functions. In addition, very few regulators of lncRNAs have been identified. We developed a novel genetic screen using reconstituted RNAi in Saccharomyces cerevisiae and systematically identified a large number of putative lncRNA repressors. Among them, we found that four highly conserved chromatin remodeling factors are global lncRNA repressors that play major roles in shaping the eukaryotic lncRNA transcriptome. Importantly, we identified >250 antisense lncRNAs (CRRATs [chromatin remodeling-repressed antisense transcripts]) whose repression by these chromatin remodeling factors is required for the maintenance of normal levels of overlapping mRNA transcripts. Our results strongly suggest that regulation of mRNA through repression of antisense lncRNAs is far more broadly used than previously appreciated.

Expansion of antisense lncRNA transcriptomes in budding yeast species since the loss of RNAi.

Antisense long noncoding RNAs (ASlncRNAs) have been implicated in regulating gene expression in response to physiological cues. However, little is known about the evolutionary dynamics of ASlncRNA and what underlies the evolution of their expression. Here, using budding yeast Saccharomyces spp. and Naumovozyma castellii as models, we show that ASlncRNA repertoires have expanded since the loss of RNA interference (RNAi), in terms of their expression levels, their lengths and their degree of overlap with coding genes. Furthermore, we show that RNAi is inhibitory to ASlncRNA transcriptomes and that increased expression of ASlncRNAs in the presence of RNAi is deleterious to N. castellii, which has retained RNAi. Together, our results suggest that the loss of RNAi had substantial effects on the genome-wide increase in expression of ASlncRNAs during the evolution of budding yeasts.

Systematic approaches to identify functional lncRNAs.

Long noncoding RNAs (lncRNAs) were discovered in eukaryotes more than 30 years ago [1]. Recent advances in genomics have led to the discovery that lncRNAs are transcribed pervasively across the genome [2(•),3,4,5(•)]. There are an increasing number of reports that identify lncRNAs whose expression is modulated during cell differentiation or in disease states. However, biological functions for the vast majority of them are yet to be determined. Here, we propose two ways to identify lncRNAs that have biological functions: to identify lncRNAs with dedicated preinitiation complexes (PICs), and to focus on those whose transcription is highly regulated.

A protein-based EM label for RNA identifies the location of exons in spliceosomes.

To locate key RNA features in the structure of the spliceosome by EM, we fused a sequence-specific RNA binding protein to a protein with a distinct donut-shaped structure. We used this fusion to label spliceosomes assembled on a pre-mRNA that contained the target sequence in the exons. The label is clearly visible in EM images of the spliceosome, and subsequent image processing with averaging shows that the exons sit close to each other in the complex. This labeling strategy will serve as a general tool for analyzing the structures of RNA-containing macromolecular complexes.

Ruthenium nitrosyls derived from polypyridine ligands with carboxamide or imine nitrogen donor(s): isoelectronic complexes with different NO photolability.

As part of our search for photoactive ruthenium nitrosyls, a set of {RuNO}6 nitrosyls has been synthesized and structurally characterized. In this set, the first nitrosyl [(SBPy3)Ru(NO)](BF4)3 (1) is derived from a polypyridine Schiff base ligand SBPy3, while the remaining three nitrosyls are derived from analogous polypyridine ligands containing either one ([(PaPy3)Ru(NO)](BF4)2 (2)) or two ([(Py3P)Ru(NO)]BF4 (3) and [(Py3P)Ru(NO)(Cl)] (4)) carboxamide group(s). The coordination structures of 1 and 2 are very similar except that in 2, a carboxamido nitrogen is coordinated to the ruthenium center in place of an imine nitrogen in case of 1. In 3 and 4, the ruthenium center is coordinated to two carboxamido nitrogens in the equatorial plane and the bound NO is trans to a pyridine nitrogen (in 3) and chloride (in 4), respectively. Complexes 1-3 contain N6 donor set, and the NO stretching frequencies (nuNO) correlate well with the N-O bond distances. All four diamagnetic {RuNO}(6) nitrosyls are photoactive and release NO rapidly upon illumination with low-intensity (5-10 mW) UV light. Interestingly, photolysis of 1 generates the diamagnetic Ru(II) photoproduct [(SBPy3)Ru(MeCN)](2+) while 2-4 afford paramagnetic Ru(III) species in MeCN solution. The quantum yield values of NO release under UV illumination (lambda(max) = 302 nm) lie in the range 0.06-0.17. Complexes 3 and 4 also exhibit considerable photoactivity under visible light. The efficiency of NO release increases in the order 2 < 3 < 4, indicating that photorelease of NO is facilitated by (a) the increase in the number of coordinated carboxamido nitrogen(s) and (b) the presence of negatively charged ligands (like chloride) trans to the bound NO.