PTBP1-activated co-transcriptional splicing controls epigenetic status of pluripotent stem cells.

Many spliceosomal introns are excised from nascent transcripts emerging from RNA polymerase II (RNA Pol II). The extent of cell-type-specific regulation and possible functions of such co-transcriptional events remain poorly understood. We examined the role of the RNA-binding protein PTBP1 in this process using an acute depletion approach followed by the analysis of chromatin- and RNA Pol II-associated transcripts. We show that PTBP1 activates the co-transcriptional excision of hundreds of introns, a surprising effect given that this protein is known to promote intron retention. Importantly, some co-transcriptionally activated introns fail to complete their splicing without PTBP1. In a striking example, retention of a PTBP1-dependent intron triggers nonsense-mediated decay of transcripts encoding DNA methyltransferase DNMT3B. We provide evidence that this regulation facilitates the natural decline in DNMT3B levels in developing neurons and protects differentiation-specific genes from ectopic methylation. Thus, PTBP1-activated co-transcriptional splicing is a widespread phenomenon mediating epigenetic control of cellular identity.

An RRM-ZnF RNA recognition module targets RBM10 to exonic sequences to promote exon exclusion.

RBM10 is an RNA-binding protein that plays an essential role in development and is frequently mutated in the context of human disease. RBM10 recognizes a diverse set of RNA motifs in introns and exons and regulates alternative splicing. However, the molecular mechanisms underlying this seemingly relaxed sequence specificity are not understood and functional studies have focused on 3΄ intronic sites only. Here, we dissect the RNA code recognized by RBM10 and relate it to the splicing regulatory function of this protein. We show that a two-domain RRM1-ZnF unit recognizes a GGA-centered motif enriched in RBM10 exonic sites with high affinity and specificity and test that the interaction with these exonic sequences promotes exon skipping. Importantly, a second RRM domain (RRM2) of RBM10 recognizes a C-rich sequence, which explains its known interaction with the intronic 3΄ site of NUMB exon 9 contributing to regulation of the Notch pathway in cancer. Together, these findings explain RBM10's broad RNA specificity and suggest that RBM10 functions as a splicing regulator using two RNA-binding units with different specificities to promote exon skipping.

Expression and clinical significance of CRABP1 and CRABP2 in non-small cell lung cancer.

The impairment of retinoic acid (RA)-dependent signaling is a frequent event during carcinogenesis. Cellular retinoic acid-binding proteins (CRABP1 and CRABP2) are important modulators of RA activity. Up to date, the role of these proteins in cancer progression remains poorly investigated. Here, we studied for the first time the simultaneous messenger RNA (mRNA) and protein expression of CRABPs in non-small cell lung cancer (NSCLC) samples. CRABP1 and CRABP2 mRNA levels were elevated in 42 and 56 % of NSCLC samples, respectively. Decrease of CRABP2 mRNA expression was significantly associated with the presence of lymph node metastases. Protein expression of CRABP1 and CRABP2 was detected in 50 and 56 % of tumor samples, respectively. We also found a positive correlation between CRABP1 and CRABP2 expression. Taken together, we demonstrated significant changes in CRABP expression in NSCLC samples. Importantly, the presented data provide the first evidence of potential involvement of CRABP2 in lung cancer metastasis.

Protocol for auxin-inducible depletion of the RNA-binding protein PTBP1 in mouse embryonic stem cells.

Inducible degradation of proteins of interest provides a powerful approach for functional studies. Here, we present a protocol for tightly controlled depletion of the RNA-binding protein PTBP1 in mouse embryonic stem cells (ESCs). We describe steps for establishing an ESC line expressing doxycycline-inducible auxin receptor protein OsTIR1 and tagging endogenous Ptbp1 alleles using CRISPR-Cas9 and homology-directed repair reagents. We then detail procedures for assaying the efficiency of inducible PTBP1 knockdown by immunoblotting. This protocol is adaptable for other protein targets. For complete details on the use and execution of this protocol, please refer to Iannone et al.1.

Conserved noncanonical residue Gly-126 confers instability to the middle part of the tropomyosin molecule.

Tropomyosin (Tm) is a two-stranded α-helical coiled-coil protein with a well established role in regulation of actin cytoskeleton and muscle contraction. It is believed that many Tm functions are enabled by its flexibility whose nature has not been completely understood. We hypothesized that the well conserved non-canonical residue Gly-126 causes local destabilization of Tm. To test this, we substituted Gly-126 in skeletal muscle α-Tm either with an Ala residue, which should stabilize the Tm α-helix, or with an Arg residue, which is expected to stabilize both α-helix and coiled-coil structure of Tm. We have shown that both mutations dramatically reduce the rate of Tm proteolysis by trypsin at Asp-133. Differential scanning calorimetry was used for detailed investigation of thermal unfolding of the Tm mutants, both free in solution and bound to F-actin. It was shown that a significant part of wild type Tm unfolds in a non-cooperative manner at low temperature, and both mutations confer cooperativity to this part of the Tm molecule. The size of the flexible middle part of Tm is estimated to be 60-70 amino acid residues, about a quarter of the Tm molecule. Thus, our results show that flexibility is unevenly distributed in the Tm molecule and achieves the highest extent in its middle part. We conclude that the highly conserved Gly-126, acting in concert with the previously identified non-canonical Asp-137, destabilizes the middle part of Tm, resulting in a more flexible region that is important for Tm function.

A transcriptome-wide antitermination mechanism sustaining identity of embryonic stem cells.

Eukaryotic gene expression relies on extensive crosstalk between transcription and RNA processing. Changes in this composite regulation network may provide an important means for shaping cell type-specific transcriptomes. Here we show that the RNA-associated protein Srrt/Ars2 sustains embryonic stem cell (ESC) identity by preventing premature termination of numerous transcripts at cryptic cleavage/polyadenylation sites in first introns. Srrt interacts with the nuclear cap-binding complex and facilitates recruitment of the spliceosome component U1 snRNP to cognate intronic positions. At least in some cases, U1 recruited in this manner inhibits downstream cleavage/polyadenylation events through a splicing-independent mechanism called telescripting. We further provide evidence that the naturally high expression of Srrt in ESCs offsets deleterious effects of retrotransposable sequences accumulating in its targets. Our work identifies Srrt as a molecular guardian of the pluripotent cell state.

Complex Selection on Human Polyadenylation Signals Revealed by Polymorphism and Divergence Data.

Polyadenylation is a step of mRNA processing which is crucial for its expression and stability. The major polyadenylation signal (PAS) represents a nucleotide hexamer that adheres to the AATAAA consensus sequence. Over a half of human genes have multiple cleavage and polyadenylation sites, resulting in a great diversity of transcripts differing in function, stability, and translational activity. Here, we use available whole-genome human polymorphism data together with data on interspecies divergence to study the patterns of selection acting on PAS hexamers. Common variants of PAS hexamers are depleted of single nucleotide polymorphisms (SNPs), and SNPs within PAS hexamers have a reduced derived allele frequency (DAF) and increased conservation, indicating prevalent negative selection; at the same time, the SNPs that "improve" the PAS (i.e., those leading to higher cleavage efficiency) have increased DAF, compared to those that "impair" it. SNPs are rarer at PAS of "unique" polyadenylation sites (one site per gene); among alternative polyadenylation sites, at the distal PAS and at exonic PAS. Similar trends were observed in DAFs and divergence between species of placental mammals. Thus, selection permits PAS mutations mainly at redundant and/or weakly functional PAS. Nevertheless, a fraction of the SNPs at PAS hexamers likely affect gene functions; in particular, some of the observed SNPs are associated with disease.

CRABP1 provides high malignancy of transformed mesenchymal cells and contributes to the pathogenesis of mesenchymal and neuroendocrine tumors.

CRABP1 (cellular retinoic acid binding protein 1) belongs to the family of fatty acid binding proteins. Retinoic acid binding is the only known functional activity of this protein. The role of CRABP1 in human carcinogenesis remains poorly understood. Here, for the first time we demonstrated pro-metastatic and pro-tumorigenic activity of CRABP1 in mesenchymal tumors. Further functional analysis revealed that the pro-tumorigenic effect of CRABP1 does not depend on retinoic acid binding activity. These results suggest that CRABP1 could have an alternative intracellular functional activity that contributes to the high malignancy of transformed mesenchymal cells. Microarray analysis detected CRABP1-mediated alterations in the expression of about 100 genes, including those encoding key regulatory proteins. CRABP1 is ubiquitously expressed in monophasic synovial sarcomas, while in biphasic synovial sarcomas it is expressed uniquely by the spindle cells of the aggressive mesenchymal component. High level of CRABP1 expression is associated with lymph node metastasis and poor differentiation/high grade of pancreatic neuroendocrine tumors (pNETs). Presented data suggest CRABP1 as a promising biomarker of pNETs' clinical behavior. Our results give the first evidence of pro-tumorigenic and pro-metastatic activity of CRABP1 in mesenchymal and neuroendocrine tumors.