Martino Colombo

Minor intron splicing is regulated by FUS and affected by ALS‐associated FUS mutants

Fused in sarcoma (FUS) is a ubiquitously expressed RNA‐binding protein proposed to function in various RNA metabolic pathways, including transcription regulation, pre‐mRNA splicing, RNA transport and microRNA processing. Mutations in the FUS gene were identified in patients with amyotrophic lateral sclerosis (ALS), but the pathomechanisms by which these mutations cause ALS are not known. Here, we show that FUS interacts with the minor spliceosome constituent U11 snRNP, binds preferentially to minor introns and directly regulates their removal. Furthermore, a FUS knockout in neuroblastoma cells strongly disturbs the splicing of minor intron‐containing mRNAs, among them mRNAs required for action potential transmission and for functional spinal motor units. Moreover, an ALS‐associated FUS mutant that forms cytoplasmic aggregates inhibits splicing of minor introns by trapping U11 and U12 snRNAs in these aggregates. Collectively, our findings suggest a possible pathomechanism for ALS in which mutated FUS inhibits correct splicing of minor introns in mRNAs encoding proteins required for motor neuron survival.

Multimodal stimulus coding by a gustatory sensory neuron in Drosophila larvae

Accurate perception of taste information is crucial for animal survival. In adult Drosophila, gustatory receptor neurons (GRNs) perceive chemical stimuli of one specific gustatory modality associated with a stereotyped behavioural response, such as aversion or attraction. We show that GRNs of Drosophila larvae employ a surprisingly different mode of gustatory information coding. Using a novel method for calcium imaging in the larval gustatory system, we identify a multimodal GRN that responds to chemicals of different taste modalities with opposing valence, such as sweet sucrose and bitter denatonium, reliant on different sensory receptors. This multimodal neuron is essential for bitter compound avoidance, and its artificial activation is sufficient to mediate aversion. However, the neuron is also essential for the integration of taste blends. Our findings support a model for taste coding in larvae, in which distinct receptor proteins mediate different responses within the same, multimodal GRN.

FUS controls the processing of snoRNAs into smaller RNA fragments that can regulate gene expression

FUS is a multifunctional protein involved in many pathways of RNA metabolism in human cells, including transcription, splicing, miRNA processing and replication-dependent histone gene expression. In this paper, we show for the first time that in human cells FUS can mediate the biogenesis of sdRNA, snoRNA-derived RNAs, that can be further involved in the regulation of gene expression. Using RNA immunoprecipitation followed by high throughput sequencing we identified snoRNAs in FUS-immunoprecipitated fraction. The interaction of FUS with a snoRNA fragment was further confirmed by EMSA and double filter binding assay. We observed that FUS negatively influences the level of selected mature snoRNAs in cells. Scanning of available human small RNAs databases revealed the existence of sdRNAs with the length of 19-33 nucleotides, that can be derived from FUS-dependent snoRNAs. Further in silico approach enabled us to predict putative targets for these sdRNAs. Our preliminary results indicate that sdRNAs may bind to the untranslated region of target mRNAs and influence their posttranscriptional stability or translation. Moreover, we identified a sdRNA that can interact with noncoding transcript and destabilize it, which in turn, might stabilize the level of mRNA transcribed from the same genomic region.

CRISPR-Trap: a clean approach for the generation of gene knockouts and gene replacements in human cells

Conventional nonhomologous end joining–based gene knockouts can lead to the production of C-terminally truncated proteins with potentially residual or dominant negative functions. Combining CRISPR/Cas9 with gene traps targeting the first intron (CRISPR-Trap), however, completely prevents the expression of the open reading frame, resulting in clean gene knockouts.

Gene expression in chronic granulomatous disease and interferon-γ receptor-deficient cells treated in vitro with interferon-γ.

Interferon‐γ (IFN‐γ) plays an important role in innate and adaptive immunity against intracellular infections and is used clinically for the prevention and control of infections in chronic granulomatous disease (CGD) and inborn defects in the IFN‐γ/interleukin (IL)‐12 axis. Using transcriptome profiling (RNA‐seq), we sought to identify differentially expressed genes, transcripts and exons in Epstein‐Barr virus–transformed B lymphocytes (B‐EBV) cells from CGD patients, IFN‐γ receptor deficiency patients, and normal controls, treated in vitro with IFN‐γ for 48 hours. Our results show that IFN‐γ increased the expression of a diverse array of genes related to different cellular programs. In cells from normal controls and CGD patients, IFN‐γ‐induced expression of genes relevant to oxidative killing, nitric oxide synthase pathway, proteasome‐mediated degradation, antigen presentation, chemoattraction, and cell adhesion. IFN‐γ also upregulated genes involved in diverse stages of messenger RNA (mRNA) processing including pre‐mRNA splicing, as well as others implicated in the folding, transport, and assembly of proteins. In particular, differential exon expression of WARS (encoding tryptophanyl‐transfer RNA synthetase, which has an essential function in protein synthesis) induced by IFN‐γ in normal and CGD cells suggests that this gene may have an important contribution to the benefits of IFN‐γ treatment for CGD. Upregulation of mRNA and protein processing related genes in CGD and IFNRD cells could mediate some of the effects of IFN‐γ treatment. These data support the concept that IFN‐γ treatment may contribute to increased immune responses against pathogens through regulation of genes important for mRNA and protein processing.

Cbp80 is needed for the expression of piRNA components and piRNAs

Cap binding protein 80 (Cbp80) is the larger subunit of the nuclear cap-binding complex (nCBC), which is known to play important roles in nuclear mRNA processing, export, stability and quality control events. Reducing Cbp80 mRNA levels in the female germline revealed that Cbp80 is also involved in defending the germline against transposable elements. Combining such knockdown experiments with large scale sequencing of small RNAs further showed that Cbp80 is involved in the initial biogenesis of piRNAs as well as in the secondary biogenesis pathway, the ping-pong amplification cycle. We further found that Cbp80 knockdown not only led to the upregulation of transposons, but also to delocalization of Piwi, Aub and Ago3, key factors in the piRNA biosynthesis pathway. Furthermore, compared to controls, levels of Piwi and Aub were also reduced upon knock down of Cbp80. On the other hand, with the same treatment we could not detect significant changes in levels or subcellular distribution (nuage localization) of piRNA precursor transcripts. This shows that Cbp80 plays an important role in the production and localization of the protein components of the piRNA pathway and it seems to be less important for the production and export of the piRNA precursor transcripts.