Carsten A. Raabe

Biases in small RNA deep sequencing data

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.

A global view of the nonprotein-coding transcriptome in Plasmodium falciparum

Nonprotein-coding RNAs (npcRNAs) represent an important class of regulatory molecules that act in many cellular pathways. Here, we describe the experimental identification and validation of the small npcRNA transcriptome of the human malaria parasite Plasmodium falciparum. We identified 630 novel npcRNA candidates. Based on sequence and structural motifs, 43 of them belong to the C/D and H/ACA-box subclasses of small nucleolar RNAs (snoRNAs) and small Cajal body-specific RNAs (scaRNAs). We further observed the exonization of a functional H/ACA snoRNA gene, which might contribute to the regulation of ribosomal protein L7a gene expression. Some of the small npcRNA candidates are from telomeric and subtelomeric repetitive regions, suggesting their potential involvement in maintaining telomeric integrity and subtelomeric gene silencing. We also detected 328 cis-encoded antisense npcRNAs (asRNAs) complementary to P. falciparum protein-coding genes of a wide range of biochemical pathways, including determinants of virulence and pathology. All cis-encoded asRNA genes tested exhibit lifecycle-specific expression profiles. For all but one of the respective sense–antisense pairs, we deduced concordant patterns of expression. Our findings have important implications for a better understanding of gene regulatory mechanisms in P. falciparum, revealing an extended and sophisticated npcRNA network that may control the expression of housekeeping genes and virulence factors.

Experimental identification and characterization of 97 novel npcRNA candidates in Salmonella enterica serovar Typhi

We experimentally identified and characterized 97 novel, non-protein-coding RNA candidates (npcRNAs) from the human pathogen Salmonella enterica serovar Typhi (hereafter referred to as S. typhi). Three were specific to S. typhi, 22 were restricted to Salmonella species and 33 were differentially expressed during S. typhi growth. We also identified Salmonella Pathogenicity Island-derived npcRNAs that might be involved in regulatory mechanisms of virulence, antibiotic resistance and pathogenic specificity of S. typhi. An in-depth characterization of S. typhi StyR-3 npcRNA showed that it specifically interacts with RamR, the transcriptional repressor of the ramA gene, which is involved in the multidrug resistance (MDR) of Salmonella. StyR-3 interfered with RamR–DNA binding activity and thus potentially plays a role in regulating ramA gene expression, resulting in the MDR phenotype. Our study also revealed a large number of cis-encoded antisense npcRNA candidates, supporting previous observations of global sense–antisense regulatory networks in bacteria. Finally, at least six of the npcRNA candidates interacted with the S. typhi Hfq protein, supporting an important role of Hfq in npcRNA networks. This study points to novel functional npcRNA candidates potentially involved in various regulatory roles including the pathogenicity of S. typhi.

Differential regulation of non-protein coding RNAs from Prader-Willi Syndrome locus

Prader-Willi Syndrome (PWS) is a neurogenetic disorder caused by the deletion of imprinted genes on the paternally inherited human chromosome 15q11-q13. This locus harbours a long non-protein-coding RNA (U-UBE3A-ATS) that contains six intron-encoded snoRNAs, including the SNORD116 and SNORD115 repetitive clusters. The 3′-region of U-UBE3A-ATS is transcribed in the cis-antisense direction to the ubiquitin-protein ligase E3A (UBE3A) gene. Deletion of the SNORD116 region causes key characteristics of PWS. There are few indications that SNORD115 might regulate serotonin receptor (5HT2C) pre-mRNA processing. Here we performed quantitative real-time expression analyses of RNAs from the PWS locus across 20 human tissues and combined it with deep-sequencing data derived from Cap Analysis of Gene Expression (CAGE-seq) libraries. We found that the expression profiles of SNORD64, SNORD107, SNORD108 and SNORD116 are similar across analyzed tissues and correlate well with SNORD116 embedded U-UBE3A-ATS exons (IPW116). Notable differences in expressions between the aforementioned RNAs and SNORD115 together with the host IPW115 and UBE3A cis-antisense exons were observed. CAGE-seq analysis revealed the presence of potential transcriptional start sites originated from the U-UBE3A-ATS spanning region. Our findings indicate novel aspects for the expression regulation in the PWS locus.

Transcription Analysis and Small Non-Protein Coding RNAs Associated with Bacterial Ribosomal Protein Operons

For decades ribosome biogenesis and translation represent key targets in the antimicrobial drug development to combat bacterial infections. Here we report a survey of various small non-protein coding (ncRNAs) associated with ribosomal protein (r-protein) operons in the bacterial pathogens S. aureus, V. cholerae, S. Typhi and M. tuberculosis. We identified four ncRNA candidates that overlap with important structural regions involved in translational feedback regulation. Most notable are the ncRNA 55 family containing the unique recognition site of the L10-(L12)4 complex that consequently might be involved in L10 operon regulation, and ncRNA StyR 337 that resembles the pseudoknot secondary structure of the S4 regulatory region. These findings potentially implicate the candidate ncRNAs in translational regulation of the corresponding operons. In total we report 28 intergenically encoded ncRNAs that map in sense orientation to 14 ribosomal protein operons and 13 cis-antisense encoded ncRNAs transcribed complementary to nine r-protein mRNAs. All ncRNA candidates were independently validated by extensive Northern blot hybridizations to account for growth-stage specific ncRNA transcription and to check ncRNA integrity. In addition we revisited the str-operon as experimental model to monitor internal initiation of transcription in the operon throughout bacterial growth by real-time PCR. Our data indicate additional facets of ribosomal protein operons transcription, and might lead to novel insights of ribosome biogenesis, as well as exploration of strategies involving differential drug development.

Genome sequence of the basal haplorrhine primate Tarsius syrichta reveals unusual insertions.

Tarsiers are phylogenetically located between the most basal strepsirrhines and the most derived anthropoid primates. While they share morphological features with both groups, they also possess uncommon primate characteristics, rendering their evolutionary history somewhat obscure. To investigate the molecular basis of such attributes, we present here a new genome assembly of the Philippine tarsier (Tarsius syrichta), and provide extended analyses of the genome and detailed history of transposable element insertion events. We describe the silencing of Alu monomers on the lineage leading to anthropoids, and recognize an unexpected abundance of long terminal repeat-derived and LINE1-mobilized transposed elements (Tarsius interspersed elements; TINEs). For the first time in mammals, we identify a complete mitochondrial genome insertion within the nuclear genome, then reveal tarsier-specific, positive gene selection and posit population size changes over time. The genomic resources and analyses presented here will aid efforts to more fully understand the ancient characteristics of primate genomes.

Alternative processing as evolutionary mechanism for the origin of novel nonprotein coding RNAs.

The evolution of new genes can ensue through either gene duplication and the neofunctionalization of one of the copies or the formation of a de novo gene from hitherto nonfunctional, neutrally evolving intergenic or intronic genomic sequences. Only very rarely are entire genes created de novo. Mostly, nonfunctional sequences are coopted as novel parts of existing genes, such as in the process of exonization whereby introns become exons through changes in splicing. Here, we report a case in which a novel nonprotein coding RNA evolved by intron-sequence recruitment into its structure. cDNAs derived from rat brain small RNAs, revealed a novel small nucleolar RNA (snoRNA) originating from one of the Snord115 copies in the rat Prader–Willi syndrome locus. We suggest that a single-point substitution in the Snord115 region led to the expression of a longer snoRNA variant, designated as L-Snord115. Cell culture and footprinting experiments confirmed that a single nucleotide substitution at Snord115 position 67 destabilized the kink-turn motif within the canonical snoRNA, while distal intronic sequences provided an alternate D-box region. The exapted sequence displays putative base pairing to 28S rRNA and mRNA targets.

Maternal transcription of non-protein coding RNAs from the PWS-critical region rescues growth retardation in mice

Prader-Willi syndrome (PWS) is a neurogenetic disorder caused by loss of paternally expressed genes on chromosome 15q11-q13. The PWS-critical region (PWScr) contains an array of non-protein coding IPW-A exons hosting intronic SNORD116 snoRNA genes. Deletion of PWScr is associated with PWS in humans and growth retardation in mice exhibiting ~15% postnatal lethality in C57BL/6 background. Here we analysed a knock-in mouse containing a 5′HPRT-LoxP-NeoR cassette (5′LoxP) inserted upstream of the PWScr. When the insertion was inherited maternally in a paternal PWScr-deletion mouse model (PWScrp−/m5′LoxP), we observed compensation of growth retardation and postnatal lethality. Genomic methylation pattern and expression of protein-coding genes remained unaltered at the PWS-locus of PWScrp−/m5′LoxP mice. Interestingly, ubiquitous Snord116 and IPW-A exon transcription from the originally silent maternal chromosome was detected. In situ hybridization indicated that PWScrp−/m5′LoxP mice expressed Snord116 in brain areas similar to wild type animals. Our results suggest that the lack of PWScr RNA expression in certain brain areas could be a primary cause of the growth retardation phenotype in mice. We propose that activation of disease-associated genes on imprinted regions could lead to general therapeutic strategies in associated diseases.

Emerging functions as host cell factors - an encyclopedia of annexin-pathogen interactions.

Abstract Emerging infectious diseases and drug-resistant infectious agents call for the development of innovative antimicrobial strategies. With pathogenicity now considered to arise from the complex and bi-directional interplay between a microbe and the host, host cell factor targeting has emerged as a promising approach that might overcome the limitations of classical antimicrobial drug development and could open up novel and efficient therapeutic strategies. Interaction with and modulation of host cell membranes is a recurrent theme in the host-microbe relationship. In this review, we provide an overview of what is currently known about the role of the Ca2+ dependent, membrane-binding annexin protein family in pathogen-host interactions, and discuss their emerging functions as host cell derived auxiliary proteins in microbe-host interactions and host cell targets.

Ancient Traces of Tailless Retropseudogenes in Therian Genomes

Transposable elements, once described by Barbara McClintock as controlling genetic units, not only occupy the largest part of our genome but are also a prominent moving force of genomic plasticity and innovation. They usually replicate and reintegrate into genomes silently, sometimes causing malfunctions or misregulations, but occasionally millions of years later, a few may evolve into new functional units. Retrotransposons make their way into the genome following reverse transcription of RNA molecules and chromosomal insertion. In therian mammals, long interspersed elements 1 (LINE1s) self-propagate but also coretropose many RNAs, including mRNAs and small RNAs that usually exhibit an oligo(A) tail. The revitalization of specific LINE1 elements in the mammalian lineage about 150 Ma parallels the rise of many other nonautonomous mobilized genomic elements. We previously identified and described hundreds of tRNA-derived retropseudogenes missing characteristic oligo(A) tails consequently termed tailless retropseudogenes. Additional analyses now revealed hundreds of thousands of tailless retropseudogenes derived from nearly all types of RNAs. We extracted 2,402 perfect tailless sequences (with discernible flanking target site duplications) originating from tRNAs, spliceosomal RNAs, 5S rRNAs, 7SK RNAs, mRNAs, and others. Interestingly, all are truncated at one or more defined positions that coincide with internal single-stranded regions. 5S ribosomal and U2 spliceosomal RNAs were analyzed in the context of mammalian phylogeny to discern the origin of the therian LINE1 retropositional system that evolved in our 150-Myr-old ancestor.