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 s mall n on- m essenger 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.

High-throughput RNA sequencing (RNA-seq) is considered a powerful tool for novel gene discovery and fine-tuned transcriptional profiling. The digital nature of RNA-seq is also believed to simplify meta-analysis and to reduce background noise associated with hybridization-based approaches. The development of multiplex sequencing enables efficient and economic parallel analysis of gene expression. In addition, RNA-seq is of particular value when low RNA expression or modest changes between samples are monitored. However, recent data uncovered severe bias in the sequencing of small non-protein coding RNA (small RNA-seq or sRNA-seq), such that the expression levels of some RNAs appeared to be artificially enhanced and others diminished or even undetectable. The use of different adapters and barcodes during ligation as well as complex RNA structures and modifications drastically influence cDNA synthesis efficacies and exemplify sources of bias in deep sequencing. In addition, variable specific RNA G/C-content is associated with unequal polymerase chain reaction amplification efficiencies. Given the central importance of RNA-seq to molecular biology and personalized medicine, we review recent findings that challenge small non-protein coding RNA-seq data and suggest approaches and precautions to overcome or minimize bias.

Small nucleolar RNAs (designated as snoRNAs in Eukarya or sRNAs in Archaea) can be grouped into H/ACA or C/D box snoRNA (sRNA) subclasses. In Eukarya, H/ACA snoRNAs assemble into a ribonucleoprotein (RNP) complex comprising four proteins: Cbf5p, Gar1p, Nop10p and Nhp2p. A homolog for the Nhp2p protein has not been identified within archaeal H/ACA RNPs thus far, while potential orthologs have been identified for the other three proteins. Nhp2p is related, particularly in the middle portion of the protein sequence, to the archaeal ribosomal protein and C/D box protein L7Ae. This finding suggests that L7Ae may be able to substitute for the Nhp2p protein in archaeal H/ACA sRNAs. By band shift assays, we have analyzed in vitro the interaction between H/ACA box sRNAs and protein L7Ae from the archaeon Archaeoglobus fulgidus. We present evidence that L7Ae forms specific complexes with three different H/ACA sRNAs, designated as Afu-4, Afu-46 and Afu-190 with an apparent K(d) ranging from 28 to 100 nM. By chemical and enzymatic probing we show that distinct bases located within bulges or loops of H/ACA sRNAs interact with the L7Ae protein. These findings are corroborated by mutational analysis of the L7Ae binding site. Thereby, the RNA motif required for L7Ae binding exhibits a structure, designated as the K-turn, which is present in all C/D box sRNAs. We also identified four H/ACA RNAs from the archaeal species Pyrococcus which exhibit the K-turn motif at a similar position in their structure. These findings suggest a triple role for L7Ae protein in Archaea, e.g. in ribosomes as well as H/ACA and C/D box sRNP biogenesis and function by binding to the K-turn motif.

Prader-Willi syndrome (PWS [MIM 176270]) is a neurogenetic disorder characterized by decreased fetal activity, muscular hypotonia, failure to thrive, short stature, obesity, mental retardation, and hypogonadotropic hypogonadism. It is caused by the loss of function of one or more imprinted, paternally expressed genes on the proximal long arm of chromosome 15. Several potential PWS mouse models involving the orthologous region on chromosome 7C exist. Based on the analysis of deletions in the mouse and gene expression in PWS patients with chromosomal translocations, a critical region (PWScr) for neonatal lethality, failure to thrive, and growth retardation was narrowed to the locus containing a cluster of neuronally expressed MBII-85 small nucleolar RNA (snoRNA) genes. Here, we report the deletion of PWScr. Mice carrying the maternally inherited allele (PWScr^m−/p+) are indistinguishable from wild-type littermates. All those with the paternally inherited allele (PWScr^m+/p−) consistently display postnatal growth retardation, with about 15% postnatal lethality in C57BL/6, but not FVB/N crosses. This is the first example in a multicellular organism of genetic deletion of a C/D box snoRNA gene resulting in a pronounced phenotype. Prader-Willi syndrome, or PWS, is a complex neurogenetic disorder and the most common genetic cause of life-threatening childhood obesity. Newborns have poor muscle tone, making suckling difficult, which leads to poor weight gain. After infancy, they experience extreme hunger, leading to obesity. Other symptoms include short stature, mental retardation, and often infertility. In PWS patients, a complex set of genes on the paternal chromosome 15 (in the PWS region) is missing or unexpressed. In an attempt to understand this disorder, various protein-coding genes in this region have been deleted in mice, but none of the resulting phenotypes consistently correlated with the human disease. This region also contains a cluster of genes that encode functional non-protein-coding RNAs. We deleted specifically the MBII-85 small nucleolar RNA (snoRNA) gene cluster on the parental mouse chromosome, which did not affect expression of any of the other snoRNA or protein-coding genes in the PWS region. These mice consistently displayed postnatal growth retardation starting from day 5 to 6, low postnatal lethality only in certain genetic backgrounds (<15%), and no adolescent obesity. Thus, this mouse model, with the deletion of a small, brain-specific non-protein-coding RNA, should prove useful for teasing out the various molecular pathologies of PWS.