Alexander Hüttenhofer

The expanding snoRNA world

In eukaryotes, the site-specific formation of the two prevalent types of rRNA modified nucleotides, 2-O-methylated nucleotides and pseudouridines, is directed by two large families of snoRNAs. These are termed box C/D and H/ACA snoRNAs, respectively, and exert their function through the formation of a canonical guide RNA duplex at the modification site. In each family, one snoRNA acts as a guide for one, or at most two modifications, through a single, or a pair of appropriate antisense elements. The two guide families now appear much larger than anticipated and their role not restricted to ribosome synthesis only. This is reflected by the recent detection of guides that can target other cellular RNAs, including snRNAs, tRNAs and possibly even mRNAs, and by the identification of scores of tissue-specific specimens in mammals. Recent characterization of homologs of eukaryotic modification guide snoRNAs in Archaea reveals the ancient origin of these non-coding RNA families and offers new perspectives as to their range of function.

RNomics: an experimental approach that identifies 201 candidates for novel, small, non‐messenger RNAs in mouse

In mouse brain cDNA libraries generated from small RNA molecules we have identified a total of 201 different expressed RNA sequences potentially encoding novel small non‐messenger RNA species (snmRNAs). Based on sequence and structural motifs, 113 of these RNAs can be assigned to the C/D box or H/ACA box subclass of small nucleolar RNAs (snoRNAs), known as guide RNAs for rRNA. While 30 RNAs represent mouse homologues of previously identified human C/D or H/ACA snoRNAs, 83 correspond to entirely novel snoRNAs. Among these, for the first time, we identified four C/D box snoRNAs and four H/ACA box snoRNAs predicted to direct modifications within U2, U4 or U6 small nuclear RNAs (snRNAs). Furthermore, 25 snoRNAs from either class lacked antisense elements for rRNAs or snRNAs. Therefore, additional snoRNA targets have to be considered. Surprisingly, six C/D box snoRNAs and one H/ACA box snoRNA were expressed exclusively in brain. Of the 88 RNAs not belonging to either snoRNA subclass, at least 26 are probably derived from truncated heterogeneous nuclear RNAs (hnRNAs) or mRNAs. Short interspersed repetitive elements (SINEs) are located on five RNA sequences and may represent rare examples of transcribed SINEs. The remaining RNA species could not as yet be assigned either to any snmRNA class or to a part of a larger hnRNA/mRNA. It is likely that at least some of the latter will represent novel, unclassified snmRNAs.

Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus fulgidus

In a specialized cDNA library from the archaeon Archaeoglobus fulgidus we have identified a total of 86 different expressed RNA sequences potentially encoding previously uncharacterized small non-messenger RNA (snmRNA) species. Ten of these RNAs resemble eukaryotic small nucleolar RNAs, which guide rRNA 2′-O-methylations (C/D-box type) and pseudouridylations (H/ACA-box type). Thereby, we identified four candidates for H/ACA small RNAs in an archaeal species that are predicted to guide a total of six rRNA pseudouridylations. Furthermore, we have verified the presence of the six predicted pseudouridines experimentally. We demonstrate that 22 snmRNAs are transcribed from a family of short tandem repeats conserved in most archaeal genomes and shown previously to be potentially involved in replicon partitioning. In addition, four snmRNAs derived from the rRNA operon of A. fulgidus were identified and shown to be generated by a splicing/processing pathway of pre-rRNAs. The remaining 50 RNAs could not be assigned to a known class of snmRNAs because of the lack of known structure and/or sequence motifs. Regarding their location on the genome, only nine were located in intergenic regions, whereas 33 were complementary to an ORF, five were overlapping an ORF, and three were derived from the sense orientation within an ORF. Our study further supports the importance of snmRNAs in all three domains of life.

Experimental RNomics: Identification of 140 Candidates for Small Non-Messenger RNAs in the Plant Arabidopsis thaliana

BACKGROUNDnGenomes from all organisms known to date express two types of RNA molecules: messenger RNAs (mRNAs), which are translated into proteins, and non-messenger RNAs, which function at the RNA level and do not serve as templates for translation.nnnRESULTSnWe have generated a specialized cDNA library from Arabidopsis thaliana to investigate the population of small non-messenger RNAs (snmRNAs) sized 50-500 nt in a plant. From this library, we identified 140 candidates for novel snmRNAs and investigated their expression, abundance, and developmental regulation. Based on conserved sequence and structure motifs, 104 snmRNA species can be assigned to novel members of known classes of RNAs (designated Class I snmRNAs), namely, small nucleolar RNAs (snoRNAs), 7SL RNA, U snRNAs, as well as a tRNA-like RNA. For the first time, 39 novel members of H/ACA box snoRNAs could be identified in a plant species. Of the remaining 36 snmRNA candidates (designated Class II snmRNAs), no sequence or structure motifs were present that would enable an assignment to a known class of RNAs. These RNAs were classified based on their location on the A. thaliana genome. From these, 29 snmRNA species located to intergenic regions, 3 located to intronic sequences of protein coding genes, and 4 snmRNA candidates were derived from annotated open reading frames. Surprisingly, 15 of the Class II snmRNA candidates were shown to be tissue-specifically expressed, while 12 are encoded by the mitochondrial or chloroplast genome.nnnCONCLUSIONSnOur study has identified 140 novel candidates for small non-messenger RNA species in the plant A. thaliana and thereby sets the stage for their functional analysis.

RNomics: identification and function of small, non-messenger RNAs

In the past few years, our knowledge about small non-mRNAs (snmRNAs) has grown exponentially. Approaches including computational and experimental RNomics have led to a plethora of novel snmRNAs, especially small nucleolar RNAs (snoRNAs). Members of this RNA class guide modification of ribosomal and spliceosomal RNAs. Novel targets for snoRNAs were identified such as tRNAs and potentially mRNAs, and several snoRNAs were shown to be tissue-specifically expressed. In addition, previously unknown classes of snmRNAs have been discovered. MicroRNAs and small interfering RNAs of about 21-23 nt, were shown to regulate gene expression by binding to mRNAs via antisense elements. Regulation of gene expression is exerted by degradation of mRNAs or translational regulation. snmRNAs play a variety of roles during regulation of gene expression. Moreover, the function of some snmRNAs known for decades, has been finally elucidated. Many other RNAs were identified by RNomics studies lacking known sequence and structure motifs. Future challenges in the field of RNomics include identification of the novel snmRNAs biological roles in the cell.

RNomics in Drosophila melanogaster: identification of 66 candidates for novel non‐messenger RNAs

By generating a specialised cDNA library from four different developmental stages of Drosophila melanogaster, we have identified 66 candidates for small non-messenger RNAs (snmRNAs) and have confirmed their expression by northern blot analysis. Thirteen of them were expressed at certain stages of D.melanogaster development, only. Thirty-five species belong to the class of small nucleolar RNAs (snoRNAs), divided into 15 members from the C/D subclass and 20 members from the H/ACA subclass, which mostly guide 2-O-methylation and pseudouridylation, respectively, of rRNA and snRNAs. These also include two outstanding C/D snoRNAs, U3 and U14, both functioning as pre-rRNA chaperones. Surprisingly, the sequence of the Drosophila U14 snoRNA reflects a major change of function of this snoRNA in Diptera relative to yeast and vertebrates. Among the 22 snmRNAs lacking known sequence and structure motifs, five were located in intergenic regions, two in introns, five in untranslated regions of mRNAs, eight were derived from open reading frames, and two were transcribed opposite to an intron. Interestingly, detection of two RNA species from this group implies that certain snmRNA species are processed from alternatively spliced pre-mRNAs. Surprisingly, a few snmRNA sequences could not be found on the published D.melanogaster genome, which might suggest that more snmRNA genes (as well as mRNAs) are hidden in unsequenced regions of the genome.

Plant snoRNA database

The Plant snoRNA database (http://www.scri.sari.ac.uk/plant_snoRNA/) provides information on small nucleolar RNAs from Arabidopsis and eighteen other plant species. Information includes sequences, expression data, methylation and pseudouridylation target modification sites, initial gene organization (polycistronic, single gene and intronic) and the number of gene variants. The Arabidopsis information is divided into box C/D and box H/ACA snoRNAs, and within each of these groups, by target sites in rRNA, snRNA or unknown. Alignments of orthologous genes and gene variants from different plant species are available for many snoRNA genes. Plant snoRNA genes have been given a standard nomenclature, designed wherever possible, to provide a consistent identity with yeast and human orthologues.

Neuronal BC1 RNA structure: evolutionary conversion of a tRNA(Ala) domain into an extended stem-loop structure.

By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brain-specific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 5 domain of BC1 RNA, derived from tRNA(Ala), does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNA(Ala) domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 3 end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA.

Blocking effects of the antiarrhythmic drug propafenone on the HERG potassium channel

Abstract. Propafenone has been shown to affect the delayed-rectifier potassium currents in cardiomyocytes of different animal models. In this study we investigated effects and mechanisms of action of propafenone on HERG potassium channels in oocytes of Xenopus laevis with the two-electrode voltage-clamp technique.Propafenone decreased the currents during voltage steps and the tail currents. The block was voltage-dependent and increased with positive going potentials (from 18% block of tail current amplitude at –40xa0mV to 69% at +40xa0mV with 100xa0µmol/l propafenone). The voltage dependence of block could be fitted with the sum of a monoexponential and a linear function. The fractional electrical distance was estimated to be δ=0.20. The block of current during the voltage step increased with time starting from a level of 83% of the control current. Propafenone accelerated the increase of current during the voltage step as well as the decay of tail currents (time constants of monoexponential fits decreased by 65% for the currents during the voltage step and by 37% for the tail currents with 100xa0µmol/l propafenone). The threshold concentration of propafenone effect was around 1xa0µmol/l and the concentration of half-maximal block (IC50) ranged between 13xa0µmol/l and 15xa0µmol/l for both current components. With high extracellular potassium concentrations, the IC50 value rose to 80xa0µmol/l. Acidification of the extracellular solution to pH 6.0 increased the IC50 value to 123xa0µmol/l, alkalization to pH 8.0 reduced it to 10xa0µmol/l and coexpression of the β-subunit minK had no statistically significant effect on the concentration dependence.In conclusion, propafenone has been found to block HERG potassium channels. The data suggest that propafenone affects the channels in the open state and give some hints for an intracellular site of action.

Identification of differentially expressed non-coding RNAs in embryonic stem cell neural differentiation

Protein-coding genes, guiding differentiation of ES cells into neural cells, have extensively been studied in the past. However, for the class of ncRNAs only the involvement of some specific microRNAs (miRNAs) has been described. Thus, to characterize the entire small non-coding RNA (ncRNA) transcriptome, involved in the differentiation of mouse ES cells into neural cells, we have generated three specialized ribonucleo-protein particle (RNP)-derived cDNA libraries, i.e. from pluripotent ES cells, neural progenitors and differentiated neural cells, respectively. By high-throughput sequencing and transcriptional profiling we identified several novel miRNAs to be involved in ES cell differentiation, as well as seven small nucleolar RNAs. In addition, expression of 7SL, 7SK and vault-2 RNAs was significantly up-regulated during ES cell differentiation. About half of ncRNA sequences from the three cDNA libraries mapped to intergenic or intragenic regions, designated as interRNAs and intraRNAs, respectively. Thereby, novel ncRNA candidates exhibited a predominant size of 18–30u2009nt, thus resembling miRNA species, but, with few exceptions, lacking canonical miRNA features. Additionally, these novel intraRNAs and interRNAs were not only found to be differentially expressed in stem-cell derivatives, but also in primary cultures of hippocampal neurons and astrocytes, strengthening their potential function in neural ES cell differentiation.