MicroRNAs (miRNAs) are small non-coding RNAs (sncRNAs) that function in post-transcriptional gene regulation through imperfect base pairing with mRNA targets which results in inhibition of translation and typically destabilization of bound transcripts. Sequence-based algorithms historically used to predict miRNA targets face inherent challenges in reliably reflecting in vivo interactions. Recent strategies have directly profiled miRNA-target interactions by crosslinking and ligation of sncRNAs to their targets within the RNA-induced silencing complex (RISC), followed by high throughput sequencing of the chimeric sncRNA:target RNAs. Despite the strength of these direct profiling approaches, standardized pipelines for effectively analyzing the resulting chimeric sncRNA:target RNA sequencing data are not readily available. Here we present SCRAP, a robust Small Chimeric RNA Analysis Pipeline for the bioinformatic processing of chimeric sncRNA:target RNA sequencing data. SCRAP consists of two parts, each of which are specifically optimized for the distinctive characteristics of chimeric small RNA sequencing reads: first, read processing and alignment and second, peak calling and annotation. We apply SCRAP to benchmark chimeric sncRNA:target RNA sequencing datasets generated by distinct molecular approaches, and compare SCRAP to existing chimeric RNA analysis pipelines. SCRAP has minimal hardware requirements, is cross-platform, and contains extensive annotation to broaden accessibility for processing small chimeric RNA sequencing data and enable insights about the targets of small non-coding RNAs in regulating diverse biological systems.

Neurons critically depend on regulated RNA localization and tight control of spatio-temporal gene expression to maintain their morphological and functional integrity. Mutations in the kinesin motor protein gene KIF1C cause Hereditary Spastic Paraplegia, an autosomal recessive disease leading to predominant degeneration of the long axons of central motoneurons. In this study we aimed to gain insight into the molecular function of KIF1C and understand how KIF1C dysfunction contributes to motoneuron degeneration. We used affinity proteomics in neuronally differentiated neuroblastoma cells (SH-SY5Y) to identify the protein complex associated with KIF1C in neuronal cells; candidate interactions were then validated by immunoprecipitation and mislocalization of putative KIF1C cargoes was studied by immunostainings. We found KIF1C to interact with all core components of the exon junction complex (EJC); expression of mutant KIF1C in neuronal cells leads to loss of the typical localization distally in neurites. Instead, EJC core components accumulate in the pericentrosomal region, here co-localizing with mutant KIF1C. These findings suggest KIF1C as a neuronal transporter of the EJC. Interestingly, the binding of KIF1C to the EJC is RNA-mediated, as treatment with RNAse prior to immunoprecipitation almost completely abolishes the interaction. Silica-based solid-phase extraction of UV-crosslinked RNA-protein complexes furthermore supports direct interaction of KIF1C with RNA, as recently also demonstrated for kinesin heavy chain. Taken together, our findings are consistent with a model where KIF1C transports mRNA in an EJC-bound and therefore transcriptionally silenced state along neurites, thus providing the missing link between the EJC and mRNA localization in neurons.

Human PRPF39 is a homolog of the yeast Prp39 and Prp42 paralogs. We have previously shown that human PRPF39 forms a homodimer that interacts with the CTD of U1C, mirroring the yeast Prp39/Prp42 heterodimer. We demonstrate here that PRPF39 knockdown in HEK293 cells affects many alternative splicing events primarily by reducing the usage of weak 5'ss. Additionally, PRPF39 preferentially binds to a GC-rich RNA, likely at the interface between its NTD and CTD. These data indicate that PRPF39 potentially recruits U1 snRNP to a weak 5' ss, serving as a previously unrecognized alternative splicing factor. We further demonstrate that human TIA1 binds to U1C through its RRM1 and RRM3+Q domains but has no significant binding to PRPF39. Finally, all three human LUC7L isoforms directly interact with U1C. These results reveal significant parallels to the yeast U1 snRNP structure and support the use of yeast U1 snRNP as a model for understanding the mechanism of human alternative splicing.

Neurogenesis is a finely tuned process, which depends on the balanced execution of expression programs that regulate cellular differentiation and proliferation. Different molecular players ranging from transcription factors to chromatin modulators control these programs. Adding to the complexity, also non-coding (nc)RNAs take part in this process. Here we analyzed the function of the long non-coding (lnc)RNA Malat1 during neural embryonic stem cell (ESC) differentiation. We find that deletion of Malat1 leads to inhibition of proliferation of neural progenitor cells (NPCs). Interestingly, this co-insides with an increase in the expression of miR-26 family members miR-26a and miR-26b in differentiating ESCs. Inactivation of miR-26a/b rescues the proliferative phenotype of Malat1 knockout (KO) cells and leads to accelerated neuronal differentiation of compound Malat1KO/mir-26KO ESCs. Together our work identifies a so far unknown interaction between Malat1 and miR-26 in the regulation of NPC proliferation and neuronal differentiation.

Each transfer RNA (tRNA) is aminoacylated (charged) with a genetic codon-specific amino acid at its 3' end. Charged tRNAs are primarily used for translation, whereas fluctuations in charged tRNA fractions are known to reflect cellular response to stress. Here we report the development of individual tRNA-acylation using PCR (i-tRAP), a convenient PCR-based method that can specifically quantify individual tRNA charging ratio. In this i-tRAP method, demethylases remove base methylations which are problematic for reverse transcription reaction, and β-elimination reaction specifically removes the 3' end of adenine residue in uncharged tRNA. Subsequent TaqMan MGB qRT-PCR can distinguish between cDNA of charged tRNA and uncharged tRNA. By using this method, we revealed that the charging ratio of tRNAGln(CUG) was changed in response to amino acid starvation and also the charging ratio of tRNAGln(CUG) in senescent cells was lower than in young cells under starvation conditions. i-tRAP can be applicable to the quantification of charging ratio of various tRNAs, and provides a simple and convenient method for analyzing tRNA charging.

In male mice, the transcription factor A MYB initiates the transcription of pachytene piRNA genes during meiosis. Here, we report that A MYB activates the transcription factor Tcfl5 produced in pachytene spermatocytes. Subsequently, A MYB and TCFL5 reciprocally reinforce their own transcription to establish a positive feedback circuit that triggers pachytene piRNA production. TCFL5 regulates the expression of genes required for piRNA maturation and promotes transcription of evolutionarily young pachytene piRNA genes, whereas A-MYB activates the transcription of older pachytene piRNA genes. Intriguingly, pachytene piRNAs from TCFL5-dependent young loci initiates the production of piRNAs from A-MYB-dependent older loci ensuring the self-propagation of pachytene piRNAs. A MYB and TCFL5 act via a set of incoherent feedforward loops that drive regulation of gene expression by pachytene piRNAs during spermatogenesis. This regulatory architecture is conserved in rhesus macaque, suggesting that it was present in the last common ancestor of placental mammals.