Mapping and characterization of macro non-protein coding RNAs in human imprinted gene regions

Genomweite Transkriptomstudien haben unterschiedliche Klassen von Nicht-Protein-kodierenden (nk) RNS offenbart und Fragen nach der Komplexität und Regulation des Genoms aufgeworfen. Genomische Prägung, ein epigenetisches Phänomen das die Exprimierung eines Gens in diploiden Zellen auf ein von zwei elterlichen Chromosomen beschränkt, ist ein Modellsystem um die Funktion der makro oder langen nkRNAs zu studieren. Sechs gut erforschte geprägte Gen- Cluster enthalten jeweils eine makro nkRNS die zwischen Maus und Mensch konserviert ist. Weiters wurde gezeigt, dass die zwei murinen makro nkRNAs Airn und Kcnq1ot1 die Exprimierung aller Protein-kodierenden Gene in den jeweiligen Gen-Clustern Igf2r und Kcnq1 unterdrücken. Beim Menschen exprimieren 8 von 27 bekannten geprägten Gen-Regionen geprägte makro nkRNAs. Um herauszufinden ob makro nkRNAs ein universelles Merkmal von allen menschlichen geprägten Gen-Regionen sind, habe ich individuell entwickelte Human Imprinted Tiling Arrays (HIRTA) und RNA-Sequenzierungstechnologien verwendet. Durch Hybridisierung von cDNA von menschlichen Geweben (20 normale und 23 Krebsgewebe) habe ich gewebespezifische Exprimierungsprofile von menschlichen geprägten Regionen erhalten. Dadurch konnte ich 101 neue Transkripte kartieren von denen 95% durch einen bioinformatische Analyse als macro nkRNS bestätigt werden konnten. Die RNA-Sequenzierung von nicht-ribosomaler RNA einer Fibroblasten Zelllinie ergab 26,2 Millionen einmalig zugeordnete Sequenzfragmente. Damit konnte ich sowohl die Exprimierung bereits bekannter geprägter makro nkRNAs erfolgreich detektieren als auch die Exprimierung von 22/23 neuen makro nkRNAs bestätigen die ich mittels HIRTA gefunden habe. Sieben neue makro nkRNAs sind entwicklungsspezifisch reguliert wie ich mittels eines Differenzierungssystems in embryonalen Stammzellen zeigen konnte. Die weitere Charakterisierung von zehn makro nkRNAs hat gezeigt, dass 4/10 ausschliesslich im Zellkern vorliegen, 6/10 monoallelisch oder bevorzugt monoallelisch exprimiert werden und 2/10 einen CpG Insel Promoter haben der unterschiedlich stark auf den beiden elterlichen Chromosomen methyliert ist (DMR). Zusammengenommen habe ich Beweise für sechs neue geprägte makro nkRNAs. Weiters sind 22 der 101 neu kartierten Transkripte nur in Krebsgeweben exprimiert. Diese könnten einen wertvollen Startpunkt für weiterführende Biomarker Forschung darstellen. Weiters konnte ich zeigen, dass alle menschlichen geprägten Gen-Regionen zumindest je eine makro nkRNA exprimieren welche geprägt sein könnte und diese daher möglicherweise eine Rolle in der Genregulation unter normalen als auch krankhaften Zuständen beim Menschen spielt.Recent genome-wide transcriptome studies revealed diverse classes of non-protein-coding (nc)RNAs and raised questions about the complexity and regulation of the genome. Genomic imprinting (an epigenetic phenomenon that restricts gene expression to one of two parental alleles in diploid cells) is a model system to study the function of an unusual class of macro or long ncRNAs. Six well-studied imprinted gene clusters contain a macro ncRNA that are mainly conserved between mouse and human. Furthermore, the mouse Airn and Kcnq1ot1 macro ncRNAs have been shown to repress all protein-coding genes, respectively in the Igf2r and Kcnq1 imprinted gene clusters. In humans, 8 out of 27 known imprinted gene regions express imprinted macro ncRNAs. To determine if macro ncRNAs are universal features of all human imprinted gene regions I used a custom Human Imprinting Region Tilling Array (HIRTA) and RNA-seq technologies. By applying 20 normal and 23 cancer human samples to HIRTA, I obtained the tissue-specific expression profiles of human imprinted gene regions and based on these profiles I mapped 101 novel transcripts of which about 95% were confirmed as macro ncRNA using a bioinformatics approach. Using ribosomal RNA depleted RNA-seq in a fibroblast cell line, I obtained 26.2 million uniquely mapped reads, successfully detected the known imprinted macro ncRNAs and validated expression of 22/23 novel macro ncRNA transcripts detected by HIRTA. 7 novel macro ncRNAs were developmentally regulated in the human embryonic stem cells differentiation system. Characterization of 10 macro ncRNAs showed that 4/10 were exclusively nuclear localized, 6/10 had monoallelic or expression biased towards one parental allele and 2/10 had CpG island promoters that are differentially methylated regions (DMRs). Thus, I have partial evidence for 6 novel imprinted macro ncRNAs. Furthermore, 22 out of the 101 mapped transcripts were expressed exclusively in cancer samples and may represent a valuable starting point for biomarkers research. In summary, all human imprinted gene regions express at least one macro ncRNA that may be imprinted and potentially play a role in gene regulation in normal or disease conditions in human

Overexpression of primary microRNA 221/222 in acute myeloid leukemia

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy with a dismal outcome in the majority of cases. A detailed understanding of the genetic alterations and gene expression changes that contribute to its pathogenesis is important to improve prognostication, disease monitoring, and therapy. In this context, leukemia-associated misexpression of microRNAs (miRNAs) has been studied, but no coherent picture has emerged yet, thus warranting further investigations. METHODS: The expression of 636 human miRNAs was compared between samples from 52 patients with AML and 13 healthy individuals by highly specific locked nucleic acid (LNA) based microarray technology. The levels of individual mature miRNAs and of primary miRNAs (pri-miRs) were determined by quantitative reverse transcriptase (qRT) PCR. Transfections and infections of human cell lines were performed using standard procedures. RESULTS: 64 miRNAs were significantly differentially expressed between AML and controls. Further studies on the clustered miRNAs 221 and 222, already known to act as oncogenes in other tumor types, revealed a deficiency of human myeloid cell lines to process vector derived precursor transcripts. Moreover, endogenous pri-miR-221/222 was overexpressed to a substantially higher extent than its mature products in most primary AML samples, indicating that its transcription was enhanced, but processing was rate limiting, in these cells. Comparison of samples from the times of diagnosis, remission, and relapse of AML demonstrated that pri-miR-221/222 levels faithfully reflected the stage of disease. CONCLUSIONS: Expression of some miRNAs is strongly regulated at the posttranscriptional level in AML. Pri-miR-221/222 represents a novel molecular marker and putative oncogene in this disease

An RNA-Seq Strategy to Detect the Complete Coding and Non-Coding Transcriptome Including Full-Length Imprinted Macro ncRNAs

Imprinted macro non-protein-coding (nc) RNAs are cis-repressor transcripts that silence multiple genes in at least three imprinted gene clusters in the mouse genome. Similar macro or long ncRNAs are abundant in the mammalian genome. Here we present the full coding and non-coding transcriptome of two mouse tissues: differentiated ES cells and fetal head using an optimized RNA-Seq strategy. The data produced is highly reproducible in different sequencing locations and is able to detect the full length of imprinted macro ncRNAs such as Airn and Kcnq1ot1, whose length ranges between 80–118 kb. Transcripts show a more uniform read coverage when RNA is fragmented with RNA hydrolysis compared with cDNA fragmentation by shearing. Irrespective of the fragmentation method, all coding and non-coding transcripts longer than 8 kb show a gradual loss of sequencing tags towards the 3′ end. Comparisons to published RNA-Seq datasets show that the strategy presented here is more efficient in detecting known functional imprinted macro ncRNAs and also indicate that standardization of RNA preparation protocols would increase the comparability of the transcriptome between different RNA-Seq datasets

Alternative Splicing and Nonsense-Mediated RNA Decay Contribute to the Regulation of SHOX Expression

The human SHOX gene is composed of seven exons and encodes a paired-related homeodomain transcription factor. SHOX mutations or deletions have been associated with different short stature syndromes implying a role in growth and bone formation. During development, SHOX is expressed in a highly specific spatiotemporal expression pattern, the underlying regulatory mechanisms of which remain largely unknown. We have analysed SHOX expression in diverse embryonic, fetal and adult human tissues and detected expression in many tissues that were not known to express SHOX before, e.g. distinct brain regions. By using RT-PCR and comparing the results with RNA-Seq data, we have identified four novel exons (exon 2a, 7-1, 7-2 and 7-3) contributing to different SHOX isoforms, and also established an expression profile for the emerging new SHOX isoforms. Interestingly, we found the exon 7 variants to be exclusively expressed in fetal neural tissues, which could argue for a specific role of these variants during brain development. A bioinformatical analysis of the three novel 3′UTR exons yielded insights into the putative role of the different 3′UTRs as targets for miRNA binding. Functional analysis revealed that inclusion of exon 2a leads to nonsense-mediated RNA decay altering SHOX expression in a tissue and time specific manner. In conclusion, SHOX expression is regulated by different mechanisms and alternative splicing coupled with nonsense-mediated RNA decay constitutes a further component that can be used to fine tune the SHOX expression level

Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA

The presence of the cap structure on the 5′-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5′-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC–MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5′ untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics

A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA

The increasing importance of in vitro-transcribed (IVT) mRNA for synthesizing the encoded therapeutic protein in vivo demands the manufacturing of pure mRNA products. The major contaminant in the IVT mRNA is double-stranded RNA (dsRNA), a transcriptional by-product that can be removed only by burdensome procedure requiring special instrumentation and generating hazardous waste. Here we present an alternative simple, fast, and cost-effective method involving only standard laboratory techniques. The purification of IVT mRNA is based on the selective binding of dsRNA to cellulose in an ethanol-containing buffer. We demonstrate that at least 90% of the dsRNA contaminants can be removed with a good, >65% recovery rate, regardless of the length, coding sequence, and nucleoside composition of the IVT mRNA. The procedure is scalable; purification of microgram or milligram amounts of IVT mRNA is achievable. Evaluating the impact of the mRNA purification in vivo in mice, increased translation could be measured for the administered transcripts, including the 1-methylpseudouridine-containing IVT mRNA, which no longer induced interferon (IFN)-α. The cellulose-based removal of dsRNA contaminants is an effective, reliable, and safe method to obtain highly pure IVT mRNA suitable for in vivo applications. Keywords: double-stranded RNA, in vitro transcription, messenger RNA, nucleoside-modified RNA, RNA purification, cellulose-based purification, RNA immunogenicit

A human haploid gene trap collection to study lncRNAs with unusual RNA biology

<p>Many thousand long non-coding (lnc) RNAs are mapped in the human genome. Time consuming studies using reverse genetic approaches by post-transcriptional knock-down or genetic modification of the locus demonstrated diverse biological functions for a few of these transcripts. The Human Gene Trap Mutant Collection in haploid KBM7 cells is a ready-to-use tool for studying protein-coding gene function. As lncRNAs show remarkable differences in RNA biology compared to protein-coding genes, it is unclear if this gene trap collection is useful for functional analysis of lncRNAs. Here we use the uncharacterized <i>LOC100288798</i> lncRNA as a model to answer this question. Using public RNA-seq data we show that <i>LOC100288798</i> is ubiquitously expressed, but inefficiently spliced. The minor spliced <i>LOC100288798</i> isoforms are exported to the cytoplasm, whereas the major unspliced isoform is nuclear localized. This shows that <i>LOC100288798</i> RNA biology differs markedly from typical mRNAs. <i>De novo</i> assembly from RNA-seq data suggests that <i>LOC100288798</i> extends 289kb beyond its annotated 3' end and overlaps the downstream <i>SLC38A4</i> gene. Three cell lines with independent gene trap insertions in <i>LOC100288798</i> were available from the KBM7 gene trap collection. RT-qPCR and RNA-seq confirmed successful lncRNA truncation and its extended length. Expression analysis from RNA-seq data shows significant deregulation of 41 protein-coding genes upon <i>LOC100288798</i> truncation. Our data shows that gene trap collections in human haploid cell lines are useful tools to study lncRNAs, and identifies the previously uncharacterized <i>LOC100288798</i> as a potential gene regulator.</p

Ribozyme Assays to Quantify the Capping Efficiency of In Vitro-Transcribed mRNA

The presence of the cap structure on the 5′-end of in vitro-transcribed (IVT) mRNA determines its translation and stability, underpinning its use in therapeutics. Both enzymatic and co-transcriptional capping may lead to incomplete positioning of the cap on newly synthesized RNA molecules. IVT mRNAs are rapidly emerging as novel biologics, including recent vaccines against COVID-19 and vaccine candidates against other infectious diseases, as well as for cancer immunotherapies and protein replacement therapies. Quality control methods necessary for the preclinical and clinical stages of development of these therapeutics are under ongoing development. Here, we described a method to assess the presence of the cap structure of IVT mRNAs. We designed a set of ribozyme assays to specifically cleave IVT mRNAs at a unique position and release 5′-end capped or uncapped cleavage products up to 30 nt long. We purified these products using silica-based columns and visualized/quantified them using denaturing polyacrylamide gel electrophoresis (PAGE) or liquid chromatography and mass spectrometry (LC–MS). Using this technology, we determined the capping efficiencies of IVT mRNAs with different features, which include: Different cap structures, diverse 5′ untranslated regions, different nucleoside modifications, and diverse lengths. Taken together, the ribozyme cleavage assays we developed are fast and reliable for the analysis of capping efficiency for research and development purposes, as well as a general quality control for mRNA-based therapeutics