THO and TRAMP complexes prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions

Expansion of structure-forming CAG/CTG repetitive sequences is the cause of several neurodegenerative disorders and deletion of repeats is a potential therapeutic strategy. Transcription-associated mechanisms are known to cause CAG repeat instability. In this study, we discovered that Thp2, an RNA export factor and member of the THO complex, and Trf4, a key component of the TRAMP complex involved in nuclear RNA degradation, are necessary to prevent CAG fragility and repeat contractions in a S. cerevisiae model system. Depletion of both Thp2 and Trf4 proteins causes a highly synergistic increase in CAG repeat fragility, indicating a complementary role of the THO and TRAMP complexes in preventing genome instability. Loss of either Thp2 or Trf4 causes an increase in RNA polymerase stalling at the CAG repeats and genome-wide transcription-replication conflicts (TRCs), implicating impairment of transcription elongation as a cause of CAG fragility and instability in their absence. Analysis of the effect of RNase H1 overexpression on CAG fragility and TRCs suggests that co-transcriptional R-loops are the main cause of CAG fragility in the thp2Δ mutants. In contrast, CAG fragility and TRCs in the trf4Δ mutant can be compensated for by RPA overexpression, suggesting that excess unprocessed RNA in TRAMP4 mutants leads to reduced RPA availability and high levels of TRCs. Our results show the importance of RNA surveillance pathways in preventing RNAPII stalling, TRCs, and DNA breaks, and show that RNA export and RNA decay factors work collaboratively to maintain genome stability.

A genetic toolkit for studying transposon control in the Drosophila melanogaster ovary

Argonaute proteins of the PIWI clade complexed with PIWI-interacting RNAs (piRNAs) protect the animal germline genome by silencing transposable elements. One of the leading experimental systems for studying piRNA biology is the Drosophila melanogaster ovary. In addition to classical mutagenesis, transgenic RNA interference (RNAi), which enables tissue-specific silencing of gene expression, plays a central role in piRNA research. Here, we establish a versatile toolkit focused on piRNA biology that combines germline transgenic RNAi, GFP marker lines for key proteins of the piRNA pathway, and reporter transgenes to establish genetic hierarchies. We compare constitutive, pan-germline RNAi with an equally potent transgenic RNAi system that is activated only after germ cell cyst formation. Stage-specific RNAi allows us to investigate the role of genes essential for germline cell survival, for example nuclear RNA export or the SUMOylation pathway, in piRNA-dependent and independent transposon silencing. Our work forms the basis for an expandable genetic toolkit provided by the Vienna Drosophila Resource Center.

A genetic toolkit for studying transposon control in the Drosophila melanogaster ovary

Argonaute proteins of the PIWI class complexed with PIWI-interacting RNAs (piRNAs) protect the animal germline genome by silencing transposable elements. One of the leading experimental systems for studying piRNA biology is the Drosophila melanogaster ovary. In addition to classical mutagenesis, transgenic RNA interference (RNAi), which enables tissue-specific silencing of gene expression, plays a central role in piRNA research. Here, we establish a versatile toolkit focused on piRNA biology that combines germline transgenic RNAi, GFP marker lines for key proteins of the piRNA pathway, and reporter transgenes to establish genetic hierarchies. We compare constitutive, pan-germline RNAi with an equally potent transgenic RNAi system that is activated only after germ cell cyst formation. Stage-specific RNAi allows us to investigate the role of genes essential for germline cell survival, for example nuclear RNA export or the SUMOylation pathway, in piRNA-dependent and independent transposon silencing. Our work forms the basis for an expandable genetic toolkit provided by the Vienna Drosophila Resource Center.

Analysis of subcellular transcriptomes by RNA proximity labeling with Halo-seq

Thousands of RNA species display nonuniform distribution within cells. However, quantification of the spatial patterns adopted by individual RNAs remains difficult, in part by a lack of quantitative tools for subcellular transcriptome analysis. In this study, we describe an RNA proximity labeling method that facilitates the quantification of subcellular RNA populations with high spatial specificity. This method, termed Halo-seq, pairs a light-activatable, radical generating small molecule with highly efficient Click chemistry to efficiently label and purify spatially defined RNA samples. We compared Halo-seq with previously reported similar methods and found that Halo-seq displayed a higher efficiency of RNA labeling, indicating that it is well suited to the investigation of small, precisely localized RNA populations. We then used Halo-seq to quantify nuclear, nucleolar, and cytoplasmic transcriptomes, characterize their dynamic nature following perturbation, and identify RNA sequence features associated with their composition. Specifically, we found that RNAs containing AU-rich elements are relatively enriched in the nucleus. This enrichment becomes stronger upon treatment with the nuclear export inhibitor leptomycin B, both expanding the role of HuR in RNA export and generating a comprehensive set of transcripts whose export from the nucleus depends on HuR.

The mammalian ecdysoneless protein interacts with RNA helicase DDX39A to regulate nuclear mRNA export

The mammalian orthologue of Ecdysoneless (ECD) protein is required for embryogenesis, cell cycle progression, and mitigation of ER stress. Here, we identified key components of the mRNA export complexes as binding partners of ECD and characterized the functional interaction of ECD with key mRNA export-related DEAD BOX protein helicase DDX39A. We find that ECD is involved in RNA export through its interaction with DDX39A. ECD knockdown (KD) blocks mRNA export from the nucleus to the cytoplasm, which is rescued by expression of full length ECD but not ECD mutant that is defective in interaction with DDX39A. We have previously shown that ECD protein is overexpressed in ErbB2+ breast cancers (BC). In this study, we extended the analyses to two publically available BC mRNA TCGA and METABRIC data sets. In both dataset, ECD mRNA overexpression correlated with short patient survival, specifically ErbB2+ BC. In METABRIC dataset ECD overexpression also correlated with poor patient survival in TNBC . Further, ECD KD in ErbB2+BC cells led to a decrease in ErbB2 mRNA level due to a block in its nuclear export, and was associated with impairment of oncogenic traits. These findings provide a novel mechanistic insight into physiological and pathological function of ECD.

Main N6-Methyladenosine Readers: YTH Family Proteins in Cancers

Among the over 150 RNA modifications, N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic RNAs, not only in messenger RNAs, but also in microRNAs and long non-coding RNAs. It is a dynamic and reversible process in mammalian cells, which is installed by “writers,” consisting of METTL3, METTL14, WTAP, RBM15/15B, and KIAA1429 and removed by “erasers,” including FTO and ALKBH5. Moreover, m6A modification is recognized by “readers,” which play the key role in executing m6A functions. IYT521-B homology (YTH) family proteins are the first identified m6A reader proteins. They were reported to participate in cancer tumorigenesis and development through regulating the metabolism of targeted RNAs, including RNA splicing, RNA export, translation, and degradation. There are many reviews about function of m6A and its role in various diseases. However, reviews only focusing on m6A readers, especially YTH family proteins are few. In this review, we systematically summarize the recent advances in structure and biological function of YTH family proteins, and their roles in human cancer and potential application in cancer therapy.

DDX41: a multifunctional DEAD-box protein involved in pre-mRNA splicing and innate immunity

DEAD-box helicases participate in nearly all steps of an RNA’s life. In recent years, increasing evidence has shown that several family members are multitasking enzymes. They are often involved in different processes, which may be typical for RNA helicases, such as RNA export and translation, or atypical, e.g., acting as nucleic acid sensors that activate downstream innate immune signaling. This review focuses on the DEAD-box protein DDX41 and summarizes our current understanding of its roles as an innate immunity sensor in the cytosol and in pre-mRNA splicing in the nucleus and discusses DDX41’s involvement in disease.