Project description:During mitosis, chromatin condensation shapes chromosomes as separate, rigid and compacted sister-chromatids to facilitate their segregation. Here, we show that unlike wild type yeast chromosomes, non-chromosomal DNA circles and chromosomes lacking a centromere fail to condense during mitosis. Genetics and ChIP-seq experiments establish that the centromere functions in chromosome condensation upstream of the kinases Aurora B and Bub1. Downstream of Aurora B and Bub1, Shugoshin and the deacetylase Hst2 facilitated spreading of the condensation signal from the pericentromeric region to the chromosome arms. Targeting Aurora B to DNA circles or centromere-ablated chromosomes, or releasing Shugoshin from PP2A-dependent inhibition bypassed the centromere requirement for condensation and enhanced the mitotic stability of DNA circles. Our data indicate that yeast cells license in a centromere-dependent manner the chromosome-autonomous condensation of their chromatin, excluding non-centromeric DNA from this process and thereby inhibiting their propagation.
Project description:We describe a case of a child affected by a relapsed PML/RARA-negative acute promyelocytic leukemia (APL) rescued by a combination of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) and subsequent HSCT. We provide evidence of the presence of a chimeric viral transcript generated through genomic integration of a Torque Teno Mini Virus sequence into RARA intron 2, the recurrent breakpoint of all chimeric RARA fusions found in APL. This generates an in-frame TTMV/RARA chimeric fusion that is highly expressed at disease diagnosis and relapse and fully cleared at remission achievement.
Project description:A substantial population of intron retentions are stably maintained in poly-adenylated transcripts and a subset of them are excised upon neuronal stimulation. These regulated intron retention events in fully transcribed RNAs represent a mechanism to rapidly mobilize a pool of mRNAs in response to neuronal activity.
Project description:Tools to understand how the spliceosome functions in vivo have lagged behind advances in its structural biology. We describe methods to globally profile spliceosome-bound precursor, intermediates and products at nucleotide resolution. We apply these tools to three divergent yeast species that span 600 million years of evolution. The sensitivity of the approach enables detection of novel cases of non- canonical catalysis including interrupted, recursive and nested splicing. Employing statistical modeling to understand the quantitative relationships between RNA features and the data, we uncover independent roles for intron size, position and number in substrate progression through the two catalytic stages. These include species-specific inputs suggestive of spliceosome-transcriptome coevolution. Further investigations reveal ATP-dependent discard of numerous endogenous substrates at both the precursor and lariat-intermediate stages and connect discard to intron retention, a form of splicing regulation. Spliceosome profiling is a quantitative, generalizable global technology to investigate an RNP central to eukaryotic gene expression.
Project description:General discard pathways eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. In contrast to such general pre-mRNA decay, we describe here a nuclear pre-mRNA degradation pathway that controls the expression of select intron-containing genes. We show that the fission yeast nuclear poly(A)-binding protein, Pab2, and the nuclear exosome subunit, Rrp6, are the main factors involved in this polyadenylation-dependent pre-mRNA degradation pathway. Transcriptome analysis and intron swapping experiments revealed that inefficient splicing is important to dictate susceptibility to Pab2-dependent pre-mRNA decay. We also show that negative splicing regulation can promote the poor splicing efficiency required for this pre-mRNA decay pathway, and in doing so identify a mechanism of cross-regulation between paralogous ribosomal proteins through nuclear pre-mRNA decay. Our findings unveil a layer of regulation in the nucleus in which the turnover of specific pre-mRNAs, besides the turnover of mature mRNAs, is used to control gene expression.
Project description:The splicing machinery associates with genes to facilitate efficient co-transcriptional mRNA processing. We have mapped these associations by genome localization analysis to ascertain how splicing is achieved and regulated on a system-wide scale. Our data show that factors important for intron recognition sample nascent mRNAs and are retained specifically at intron-containing genes via RNA-dependent interactions. Spliceosome assembly proceeds co-transcriptionally, but completes post-transcriptionally in most cases. Some intron-containing genes were not bound by the spliceosome, including several developmentally regulated genes. On this basis we predicted and verified regulated splicing, and observed a role for nuclear mRNA surveillance in monitoring those events. Finally, we present evidence that co-transcriptional processing events determine the recruitment of specific mRNA export factors. Broadly, our results provide mechanistic insights into the coordinated regulation of transcription, mRNA processing, and nuclear export in executing complex gene expression programs. Keywords: ChIP-chip
Project description:5’ tRNA-fMet1 half is delivered into cystic fibrosis airway epithelium cells by outer membrane vesicles and regulate gene expression. We used chimeric eCLIP to map transcriptome-wide interactions between 5’ tRNA-fMet1 half and target mRNAs mediated by AGO2.