Alternative miRNA functions
microRNAs are important post-transcriptional regulators. Each miRNA can target hundreds of target mRNAs. Some strongly down-regulate their targets during dynamic processes such as development. However, the functions of miRNAs in non-dynamic conditions are not well understood and many deeply conserved miRNAs only have subtle effects on their target transcripts. We investigate two hypothetical alternative miRNA functions. miRNA may buffer variations in gene expression or induce important temporal covariances between target genes. To investigate these hypothesis we make use of state-of-the-art single-cell sequencing technology.
Alternative miRNA functions : miRNA have been suggested to buffer gene expression variation. If this holds true, a broad gene expression profile (left) would be narrowed (right) through the action of targeting miRNA (center).
Gene co-variances have long been used to infer gene function and regulation. In a simple scenario TWO CONDITIONS (e.g. a disease and a healthy condition) are compared and differentially expressed genes are linked to a common function or regulatory circuit. In time-scale or dose-dependent experiments (MULTIPLE CONDITIONS) co-variances can be determined with better statistical support (e.g. when sampling different time points in C. elegans development). Single-cell sequencing allows for the inference of gene co-variances in homogeneous cell populations (ONE CONDITION) with high confidence providing a more general and unbiased view. We aim to identify regulatory layers that contribute to global covariance patterns.
Gene covariances : Gene covariances can be determined from data sets representing two or more conditions (top panels). By the means of single-cell sequencing covariances can be inferred from different cells in a homogeneous population – “one condition” – as well (bottom panels).
miRTrace is a new quality control and taxonomic tracing tool developed specifically for small RNA sequencing data (sRNA-Seq). Each sample is characterized by profiling sequencing quality, read length, sequencing depth and miRNA complexity and also the amounts of miRNAs versus undesirable sequences (derived from tRNAs, rRNAs and sequencing artifacts). In addition to these routine quality control (QC) analyses, miRTrace can accurately and sensitively resolve taxonomic origins of small RNA-Seq data based on the composition of clade-specific miRNAs. This feature can be used to detect cross-clade contaminations in typical lab settings. It can also be applied for more specific applications in forensics, food quality control and clinical diagnosis, for instance tracing the origins of meat products or detecting parasitic microRNAs in host serum.
Characterisation of small RNAs arising at DNA double strand breaks
DNA double strand breaks (DSBs) are probably the most consequential genomic assaults a cell can experience and might lead to cell cycle arrest or, in the worst case, cell death. Therefore it is of utmost importance that DSBs are quickly and efficiently repaired. Despite centuries of research it only recently became apparent that RNA is transcribed at the break and it has been suggested that it is processed into miRNA-like small RNAs (termed damage-induced small RNAs or diRNAs), utilising a similar biogenesis pathway as miRNAs. However, the way of production, processing and biological relevance of diRNAs remains obscure. We will investigate the contribution of miRNA processing factors like Drosha and Dicer to the production of diRNAs by analysing the small RNA population in wild-type and mutant cells where DSBs were induced at specific genomic loci.
DNA double strand breaks induces transcription from the break site. Using cells mutant for small RNA biogenesis factors we investigate the processing of these transcripts into damage induced small RNAs (diRNAs) via small RNA sequencing.
Quantification of miRNAs in single cells
Together with the observed effects of miRNAs on their targets in single cells it is of immense importance to quantify the number of miRNA molecules itself in each single cell. This will enable us to measure how strong or weak the effect of miRNA regulation on particular targets really is. In collaboration with Omid Faridani and Rickard Sandberg we apply and improve current state of the art single cell small RNA sequencing technologies to asses miRNA expression heterogeneity which enables quantitative statements of miRNA specific regulation. Further, the understanding of miRNA arm selection, untemplated nucleotide additions, RNA editing and SNPs in miRNA genes will be highly improved.
Single-cell sequencing is used for absolute quantification of miRNA molecules per cell.
Structure and sequence features determining miRNA biogenesis
The human transcriptome contains more than 100,000 hairpin RNA structures, more than half of which are located in mRNAs. These hairpins are potential entry points into miRNA biogenesis, and thus constitute cross-roads for nuclear transcripts. They can either be cleaved into regulatory miRNAs or they can avoid cleavage and function as full-length transcripts, for instance through cytoplasmic transport and translation. However, currently our understanding of the features that determine this critical decision is incomplete. We have developed the first method to test cleavage of thousands of truly distinct hairpins in a single screen, and we are applying computational approaches to extract new structure and sequence features which license or block the miRNA biogenesis.
Transcripts containing RNA hairpins are either cleaved to miRNAs or function as full-length transcripts, for instance by cytoplasmic export and translation. It is not fully understood what determines this critical decision.