Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of circPCMTD1 in BCR/ABL1-positive myeloid leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR/ABL1 translocations. RNA sequencing and mass cytometry experiments identified aberrant activation of the DNA damage response (DDR) pathway as a downstream effect of circPCMTD1 depletion. DNA fiber assays, Comet assays and profiling of DDR markers (phospho-H2AX, phospho-CHK1, etc.) further underscored the pronounced effect of circPCMTD1 depletion in increasing genotoxic stress and inhibiting leukemic cell growth. circPCMTD1 targeting also led to aberrant DDR activation in leukemia patient blasts with BCR/ABL1 translocations. In in vivo experiments, circPCMTD1 knock-down prolonged the survival of mice engrafted with BCR/ABL1-positive leukemia cells. Mechanistically, we found that circPCMTD1 is enriched in the cytoplasm and associates with the ribosomes of leukemic blasts. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 generates a 127 amino-acid peptide product (cPCMTD1-127aa). Using a custom-produced antibody, we found that the cPCMTD1-127aa interacts with the BCR/ABL1 oncoprotein, as well as with the BLM, TOP3A and RMI1 proteins, which form the BTR complex and regulate DNA repair and genome stability. cPCMTD1-127aa enhanced BTR complex formation, thereby increasing cellular tolerance to genotoxic stress. In summary, we identify and characterize circPCMTD1 as a molecular vulnerability and potential therapeutic target in BCR/ABL1-positive leukemias.

The RNA lariat debranching enzyme DBR1 is essential for intron turnover and RNA metabolism, yet its broader impact on transcriptome regulation remains incompletely defined. To elucidate the consequences of DBR1 depletion, we performed transcriptome-wide RNA sequencing of DBR1-knockdown and wild-type HEK293 cells. Differential expression analysis revealed widespread perturbations in pathways linked to RNA splicing, mRNA surveillance, translational control, and stress-granule biology. Many of the most significantly altered transcripts encode splicing factors and RNA quality-control components, underscoring DBR1's influence on post-transcriptional regulation. Alternative splicing analysis showed changes across multiple event types, with exon skipping accounting for >50% of events, followed by mutually exclusive exons, alternative 5' and 3' splice sites, and retained introns, indicating that DBR1 depletion induces pervasive splicing defects. Direct spliceosome inhibition using isoginkgetin (blocks tri-snRNP recruitment) and pladienolide B (targets SF3B1) reproduced the DBR1-KD mis-splicing patterns of cell signaling genes and factors involved in RNA metabolism, supporting a functional link between DBR1 activity and alternative splicing. Notably, DBR1 knockdown revealed a subset of transcripts that are both NMD-sensitive and enriched within stress granules. Consistent with this observation, G3BP1 immunopurification and confocal microscopy further support a role for DBR1 and UPF1 in stress-granule dynamics, suggesting that these factors may participate at distinct stages to influence mRNA fate under stress conditions. Together, these findings indicate that DBR1 functions beyond lariat RNA turnover as a common regulator of RNA processing, transcriptome stability, and stress granule homeostasis, revealing intricate crosstalk between RNA splicing and RNA quality control pathways in human cells.

Splicing factors are frequently mutated in myeloid cancers, causing splicing aberrations that derail the expression of tumor suppressor genes. In SRSF2 mutated cancers, a key oncogenic splicing event is the inclusion of a “poison” exon that introduces an early stop codon in EZH2 mRNA, causing its destabilization. In this issue of Genes & Development , Islam et al. (doi:10.1101/gad.353628.126) define how mutant SRSF2 binding to the poison exon mediates its inclusion and identify an antisense oligonucleotide that represses the exon to restore EZH2 function and rescues hematopoietic defects. Thus, targeting of poison exons, many of which show protumorigenic and antitumorigenic properties, is a promising new avenue to treat cancer.

Sm-ring assembly is important for the biogenesis, stability, and function of uridine-rich small nuclear RNAs (U snRNAs) involved in pre-messenger RNA (mRNA) splicing and histone pre-mRNA processing. Sm-ring assembly is cytoplasmic and dependent upon the Sm-site sequence and structural motif, ATP, and Survival motor neuron (SMN) protein complex. While RNAs other than U snRNAs were previously shown to associate with Sm proteins, whether this association follows Sm-ring assembly requirements is unknown. We systematically identified Sm-sites within the human and mouse transcriptomes and assessed whether these sites can accept Sm-rings. In addition to snRNAs, Sm-sites are highly prevalent in the 3' untranslated regions of long mRNAs. RNA immunoprecipitation experiments confirm that Sm-site containing mRNAs associate with Sm proteins in the cytoplasm. In modified Sm-ring assembly assays, Sm-site containing mRNAs, specifically associate with Sm proteins in an Sm-site, SMN, and ATP-dependent manner. In cell and animal models of Spinal Muscular Atrophy (SMA), mRNAs containing Sm-sites are downregulated, suggesting reduced Sm-ring assembly on these mRNAs may contribute to SMA pathogenesis. Together, this study establishes that Sm-site containing mRNAs can accept Sm-rings and identifies a novel mechanism for Sm proteins in regulation of cytoplasmic mRNAs.

The Exon Junction Complex (EJC) decorates RNA exon-exon junctions and modulates mRNA fate at multiple post-transcriptional steps until its disassembly during translation. Our investigation of the EJC disassembly factor PYM1 in human embryonic kidney 293 (HEK293) cells show that the EJC-PYM1 interaction is required for translation-independent EJC destabilization but not for translation-dependent disassembly. Surprisingly, PYM1 interaction deficient EJCs are enriched on locations away from canonical EJC binding site, particularly on transcripts with no or few introns. Such non-canonical EJCs are capable of inducing nonsense-mediated mRNA decay when present downstream of stop codons. Suppression of PYM1 in human cells, including by previously reported PYM1-flavivirus capsid protein interaction, stabilizes mRNAs with fewer and longer exons that localize to endoplasmic reticulum associated TIS-granules. In summary, PYM1 limits non-canonical EJC and thereby acts as an EJC specificity factor that is hijacked by flaviviruses to reshape host cell mRNA regulation.

Pre-mRNA splicing, carried out in the nucleus by a large ribonucleoprotein machine known as the spliceosome, is functionally and physically coupled to the mRNA surveillance pathway in the cytoplasm called nonsense-mediated mRNA decay (NMD). The NMD pathway monitors for premature translation termination, which can result from alternative splicing, by relying on the exon junction complex (EJC) deposited on exon-exon junctions by the spliceosome. Recently, multiple genetic screens in human cell lines have identified numerous spliceosome components as putative NMD factors. Using publicly available RNA-seq datasets from K562 and HepG2 cells depleted of 18 different spliceosome components, we found that natural NMD-targeted mRNA isoforms were upregulated when catalytic spliceosome members were reduced. While some of this increase could be due to widespread pleiotropic effects of spliceosome dysfunction (e.g. reduced expression of NMD factors due to missplicing of their mRNAs), we identified that AQR, SF3B1, SF3B4, and CDC40 may have a more direct role in NMD. We also tested the hypothesis that increased production of novel NMD substrates may overwhelm the pathway to find a direct correlation between the amount of novel NMD substrates detected and the degree of NMD inhibition observed. Finally, similar transcriptome alterations and NMD substrate upregulation were observed in cells treated with spliceosome inhibitors and in cells derived from retinitis pigmentosa patients with mutations in PRPF8 and PRPF31. Overall, our results show that regardless of the cause, spliceosome disruption upregulates a broad set of NMD targets, which could contribute to cellular dysfunction in spliceosomopathies.

Circular RNAs are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. Herein, we report on the functional relevance of the circPCMTD1 transcript in acute leukemias. In screening experiments, we found that circPCMTD1 depletion strongly inhibited the proliferative capacity of leukemic cells with BCR-ABL translocations. Mass cytometry experiments identified the aberrant activation of the DNA damage response as an early downstream event of circPCMTD1 depletion. In in vivo experiments, circPCMTD1 targeting prolonged the survival of mice engrafted with leukemic blasts harboring the Philadelphia chromosome. Mechanistically, we found that circPCMTD1 was enriched in the cytoplasm and associated with the ribosomes of the leukemic cells. We detected a cryptic open reading frame within the circPCMTD1 sequence and found that circPCMTD1 could generate a peptide product. The circPCMTD 1-derived peptide interacted with proteins of the BTR complex and enhanced BTR complex formation, thereby increasing tolerance to genotoxic stress.

Circular RNAs (circRNAs) are a novel class of RNA transcripts, which regulate important cellular functions in health and disease. CircRNAs are covalently joined and characterized by the perturbed arrangement of exons known as back-splicing. Initially regarded as transcriptional byproducts, circRNAs have been shown to regulate mRNA translation by acting as microRNA sponges, and recent studies have revealed their roles in transcription, translation, and various cellular functions. In cancer, circRNAs can function as oncogenes or tumor suppressors, and their stability makes them potential biomarkers for disease. In acute leukemias, circRNAs generated from recurrent chromosomal translocations contribute to leukemogenesis. Here, we investigate the role of circPCMTD1 in chronic myeloid leukemia (CML) in the blast crisis (BC). Functional studies using LNA-modified, RNase H-recruiting oligonucleotides (gapmers) targeting circPCMTD1 demonstrated a significant decrease in proliferation and a potent G2/M cell cycle blockade in CML-BC cell lines (K-562 & LAMA-84), both harboring the t(9;22)(q34;q11.2). Quantitative real-time PCR confirmed the specificity of circPCMTD1 depletion without affecting the linear PCMTD1 transcript. CircPCMTD1 knockdown (KD) reduced the viability of leukemic blasts, indicating its essential role in cell survival. RNA sequencing after circPCMTD1-KD in K-562 cells identified approximately 150 differentially expressed genes involved in cell cycle control, nuclear organization, and transcriptional regulation, such as SMARCA4, MACM, PCLAF, and SASH1. Gene Set Enrichment analysis highlighted rRNA processing and DNA replication-dependent chromatin function to be notably affected by circPCMTD1 depletion. CyTOF-based cell cycle analysis validated the G2/M blockade. Increased γH2AX levels indicated aberrant DNA damage response, confirmed by western blotting and intracellular flow cytometry. In addition, circPCMTD1-KD led to an increase in the phosphorylation of the CHK1, RPA32, ATR, ATM, and DNA-PK proteins. DNA fiber assays and comet assays further confirmed reduced DNA replication capacity and increased double-stranded DNA breaks upon circPCMTD1 depletion. Taken together, these data underscore the aberrant DNA damage response and the significant increase in genotoxic stress that is triggered by circPCMTD1 depletion. We performed targeted circPCMTD1 profiling in CML patients in the chronic, accelerated, and blast crisis phase and found an increased abundance of circPCMTD1 in advanced disease stages, indicating a potential role of higher circPCMTD1 expression in disease progression. In vitro experiments with patient blasts showed that circPCMTD1-KD increased γH2AX levels specifically in BCR::ABL-positive samples. In vivo, targeting circPCMTD1 in mice engrafted with BCR::ABL-positive blasts prolonged survival significantly, with no notable toxicities observed. Mechanistically, circPCMTD1 was enriched in the cytoplasm and associated with ribosomes. Polysome profiling suggested its protein-coding capacity, and we identified a cryptic open reading frame within circPCMTD1. Using custom antibodies, we detected a circPCMTD1-derived peptide (~30 KD) localized mainly in the nucleus. Immunoprecipitation followed by mass spectrometry revealed that the peptide interacted with BLM, TOP3A, and RMI1 proteins of the BTR complex. CircPCMTD1 knockdown reduced BTR complex formation. Knockdown of these proteins individually reduced leukemic blast viability, but concomitant depletion mimicked the G2/M blockade seen with circPCMTD1 depletion. Furthermore, treatment with Dasatinib, a tyrosine kinase inhibitor, decreased circPCMTD1-derived peptide levels without affecting the expression levels of the circPCMTD1 transcript and reduced BTR complex formation, linking BCR::ABL activity to circPCMTD1 function. In summary, we identify circPCMTD1 as a crucial regulator in BCR::ABL-positive leukemias, affecting DNA damage response, proliferation, and cell cycle progression. Our findings highlight circPCMTD1 as a potential therapeutic target in myeloid malignancies with t(9;22). Future studies should explore the therapeutic implications of targeting circPCMTD1 in combination with existing treatments, potentially offering a novel approach to managing drug resistance and improving outcomes in CML patients.

: 1 Introns are found in 5’ untranslated regions (5’UTRs) for 35% of all human transcripts. 2 These 5’UTR introns are not randomly distributed: genes that encode secreted, membrane- 3 bound and mitochondrial proteins are less likely to have them. Curiously, transcripts lacking 4 5’UTR introns tend to harbor specific RNA sequence elements in their early coding regions. To 5 model and understand the connection between coding-region sequence and 5’UTR intron 6 status, we developed a classifier that can predict 5’UTR intron status with >80% accuracy 7 using only sequence features in the early coding region. Thus, the classifier identifies 8 transcripts with 5’ proximal-intron-minus-like-coding regions (“5IM” transcripts). 9 Unexpectedly, we found that the early coding sequence features defining 5IM transcripts are 10 widespread, appearing in 21% of all human RefSeq transcripts. The 5IM class of transcripts is 11 enriched for non-AUG start codons, more extensive secondary structure both preceding the 12 start codon and near the 5’ cap, greater dependence on eIF4E for translation, and association 13 with ER-proximal ribosomes. 5IM transcripts are bound by the Exon Junction Complex (EJC) at 14 non-canonical 5’ proximal positions. Finally, N 1 -methyladenosines are specifically enriched in 15 the early coding regions of 5IM transcripts. Taken together, our analyses point to the existence 16 of a distinct 5IM class comprising ~20% of human transcripts. This class is defined by depletion of 5’ proximal introns, presence of specific RNA sequence features associated with 18 low translation efficiency, N 1 -methyladenosines in the early coding region, and enrichment for 19 non-canonical binding by the Exon Junction Complex. the of a distinct ‘5IM’ class comprising of transcripts. of 5’ introns, presence specific RNA sequence translation efficiency,

Nonsense-mediated mRNA decay (NMD) is a quality control pathway in eukaryotes that continuously monitors mRNA transcripts to ensure truncated polypeptides are not produced. The expression of many normal mRNAs that encode full-length polypeptides is also regulated by this pathway. Such transcript surveillance by NMD is intimately linked to translation termination. When a ribosome terminates translation at a normal termination codon, NMD is not activated, and mRNA can undergo repeated rounds of translation. On the other hand, when translation termination is deemed abnormal, such as that on a premature termination codon, it leads to a series of poorly understood events involving the NMD pathway, which destabilizes the transcript. In this review, we summarize our current understanding of how the NMD machinery interfaces with the translation termination factors to initiate NMD. We also discuss a variety of cis-acting sequence contexts and transacting factors that can cause readthrough, ribosome reinitiation, or ribosome frameshifting at stop codons predicted to induce NMD. These alternative outcomes can lead to the ribosome translating downstream of such stop codons and hence the transcript escaping NMD. NMD escape via these mechanisms can have wide-ranging implications on human health, from being exploited by viruses to hijack host cell systems to being harnessed as potential therapeutic possibilities to treat genetic diseases.

Nonsense-mediated mRNA decay (NMD) is governed by the three conserved factors-UPF1, UPF2, and UPF3. While all three are required for NMD in yeast, UPF3B is dispensable for NMD in mammals, and its paralog UPF3A is suggested to only weakly activate or even repress NMD due to its weaker binding to the exon junction complex (EJC). Here, we characterize the UPF3A/B-dependence of NMD in human cell lines deleted of one or both UPF3 paralogs. We show that in human colorectal cancer HCT116 cells, NMD can operate in a UPF3B-dependent and -independent manner. While UPF3A is almost dispensable for NMD in wild-type cells, it strongly activates NMD in cells lacking UPF3B. Notably, NMD remains partially active in cells lacking both UPF3 paralogs. Complementation studies in these cells show that EJC-binding domain of UPF3 paralogs is dispensable for NMD. Instead, the conserved "mid" domain of UPF3 paralogs is consequential for their NMD activity. Altogether, our results demonstrate that the mammalian UPF3 proteins play a more active role in NMD than simply bridging the EJC and the UPF complex.

Nonsense-mediated mRNA decay (NMD) is a conserved translation-coupled quality control mechanism in all eukaryotes that regulates the expression of a significant fraction of both the aberrant and normal transcriptomes. In vertebrates, NMD has become an essential process owing to expansion of the diversity of NMD-regulated transcripts, particularly during various developmental processes. Surprisingly, however, some core NMD factors that are essential for NMD in simpler organisms appear to be dispensable for vertebrate NMD. At the same time, numerous NMD enhancers and suppressors have been identified in multicellular organisms including vertebrates. Collectively, the available data suggest that vertebrate NMD is a complex, branched pathway wherein individual branches regulate specific mRNA subsets to fulfill distinct physiological functions.

RNA-binding proteins (RBPs) regulate all aspects of RNA metabolism. The ability to identify RNA targets bound by RBPs is critical for understanding RBP function. While powerful techniques are available to identify binding sites of individual RBPs at high resolution, it remains challenging to unravel binding sites of multicomponent ribonucleoproteins (RNPs) where multiple RBPs or proteins function cooperatively to bind to target RNAs. To fill this gap, we have previously developed RNA Immunoprecipitation in Tandem followed by high-throughput sequencing (RIPiT-seq) to characterize RNA targets of compositionally distinct RNP complexes by sequentially immunoprecipitating two proteins from the same RNP and sequencing the co-purifying RNA footprints. Here, we provide an updated and improved protocol for RIPiT-seq. In this protocol, we have used CRISPR-Cas9 to introduce affinity tag to endogenous protein of interest to capture a more representative state of an RNP complex. We present a modified protocol for library preparation for high-throughput sequencing so that it exclusively uses equipment and reagents available in a standard molecular biology lab. This updated custom library preparation protocol is compatible with commercial PCR multiplexing systems for Illumina sequencing platform for simultaneous and cost-effective analysis of large number of samples.

ABSTRACTNonsense-mediated mRNA decay (NMD) is governed by the three conserved factors - UPF1, UPF2 and UPF3. While all three are required for NMD in yeast, UPF3B is dispensable for NMD in mammals, with its paralog UPF3A suggested to only weakly activate or even repress NMD due to its weaker binding to the exon junction complex (EJC). Here we characterize the UPF3B-dependent and -independent NMD in human cell lines knocked-out of one or bothUPF3paralogs. We show that in human colorectal cancer HCT116 cells, EJC-mediated NMD can operate in UPF3B-dependent and -independent manner. While UPF3A is almost completely dispensable for NMD in wild-type cells, it strongly activates EJC-mediated NMD in cells lacking UPF3B. Surprisingly, this major NMD branch can operate in UPF3-independent manner questioning the idea that UPF3 is needed to bridge UPF proteins to the EJC during NMD. Complementation studies in UPF3 knockout cells further show that EJC-binding domain of UPF3 paralogs is not essential for NMD. Instead, the conserved mid domain of UPF3B, previously shown to engage with ribosome release factors, is required for its full NMD activity. Altogether, UPF3 plays a more active role in NMD than simply being a bridge between the EJC and the UPF complex.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Throughout their lifetime, messenger RNAs (mRNA) exist decorated with proteins as mRNA‐protein particles, or mRNPs. A key component of all spliced mRNPs is the exon junction complex (EJC), which assembles during pre‐mRNA splicing ~24 nucleotides (nt) upstream of exon‐exon junctions. The stable EJC core thus assembled serves as an interaction platform for peripheral proteins that direct mRNA export, localization, translation and nonsense‐mediated mRNA decay (NMD). Both mRNPs and EJCs and are widely presumed to be "dynamic" entities that change during an mRNAs lifetime. We recently discovered that EJCs, and hence spliced mRNPs, undergo an extensive compositional overhaul after their export to cytoplasm. This compositional switch has important implications for the EJC‐dependent NMD pathway, which can now be divided into at least two distinct phases. We find that two compositionally distinct EJCs differentially associate with the NMD factor UPF3B, which is also a peripheral EJC factor that is required for efficient NMD of only a small subset of mRNAs. We will discuss new insights gained from our work into the mechanism of EJC‐dependent NMD in the absence of UPF3B, and about the function of UPF3B in NMD. Overall, our work is shedding new light on how mRNP composition specifies distinct phases or branches of the NMD pathway, an essential post‐transcriptional mechanism that controls cellular adaptation, differentiation and development.Support or Funding InformationThis work is supported by NIH (R01 GM120209). ZY is supported by graduate fellowships from Pelotonia and OSU Center for RNA Biology.

Repository Citation Cenik C, Chua HN, Singh G, Akef A, Snyder MP, Palazzo AF, Moore MJ, Roth FP. (2016). A Common Class of Transcripts with 5'-Intron Depletion, Distinct Early Coding Sequence Features, and N1-Methyladenosine Modification [preprint]. University of Massachusetts Medical School Faculty Publications. https://doi.org/ 10.1101/057455. Retrieved from https://escholarship.umassmed.edu/faculty_pubs/1561