Project description:RNA helicases are critical regulators of gene expression and therefore strict regulation of their activities is essential to limit promiscuity and ensure target specify. While some RNA helicases appear dedicated for particular substrates others, such as DHX15, are multifunctional, contributing to more than one gene expression process. Protein cofactors play key roles both in modulating RNA helicase activities and also directing them to appropriate substrate RNAs. The largest family of RNA helicase cofactors, with more than 20 members expressed in human cells, are the G-patch proteins, characterised by a glycine-rich domain via which they associate with specific DEAH/RHA helicases. However, the cognate RNA helicases and cellular functions of numerous human G-patch proteins still remain elusive. Here, we reveal that the G-patch protein GPATCH4 is a cofactor of DHX15 that stimulates its ATPase activity. We show that GPATCH4 interacts with DHX15 in the nucleolus via residues in its G-patch domain and co-migrates with pre-ribosomal particles, wherein loss of DHX15 reduces pre-ribosomal co-migration of GPATCH4. Using a combination of high-throughput sequencing approaches (ChIP, CRAC and small RNA-seq), we discovered that GPATCH4 crosslinks to the ribosomal DNA locus and precursor ribosomal RNAs as well as binding to small nucleolar- and small Cajal body-associated- RNAs that guide rRNA and snRNA modifications. In line with this, using RiboMeth-seq analysis to globally analyse rRNA and snRNA 2’-O-methylation revealed that knockout of GPATCH4 impairs methylation at various sites. Utilizing targeted 2’-O-methylation-sensitive RNase H-based cleavage assays, we demonstrated that the regulation of 2’-O-methylation by GPATCH4 is both dependent on, and independent of, its interaction with DHX15. Intriguingly, the ATPase activity of DHX15 is necessary for efficient methylation of DHX15-dependent sites, suggesting a function of DHX15 in regulating snoRNA-guided 2’-O-methylation of rRNA that requires activation by GPATCH4. Overall, our findings not only extend knowledge on RNA helicase regulation by G-patch proteins but also provide important new insights into how the installation of rRNA and snRNA modifications, which are essential for ribosome assembly/function and pre-mRNA splicing, is regulated.

Project description:Fine-tuning of osteoclast differentiation (OD) and bone remodeling is crucial for bone homeostasis. Dissecting the mechanisms regulating osteoclastogenesis is fundamental to understanding the pathogenesis of various bone disorders including osteoporosis and arthritis. Nuclear factor erythroid 2-related factor 1 (NFE2L1, also known as NRF1), which belongs to the CNC-bZIP family of transcription factors, orchestrates a variety of physiological processes and stress responses. While Nfe2l1 gene may be transcribed into multiple alternatively spliced isoforms, the biological function of the different isoforms of NFE2L1 in bone metabolism, osteoclastogenesis in particular, has not been reported. Here we demonstrate that knockout of all isoforms of Nfe2l1 transcripts specifically in the myeloid lineage in mice [Nfe2l1(M)-KO] results in increased activity of osteoclasts, decreased bone mass and worsening of osteoporosis induced by ovariectomy and aging. In comparison, LysM-Cre-mediated Nfe2l1 deletion has no significant effect on the osteoblast and osteocytes. Mechanistic investigations using bone marrow cells and RAW 264.7 cells revealed that deficiency of Nfe2l1 leads to accelerated and elevated OD, which is attributed, at least in part, to enhanced accumulation of ROS in the early stage of OD and expression of nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 1α (Nfatc1/α). In addition, NFE2L1 regulates the transcription of multiple antioxidant genes and Nfatc1/α and OD in an isoform-specific manner. While long isoforms of NFE2L1 function as accelerators of induction of Nfatc1/α and antioxidant genes and OD, the short isoform NFE2L1-453 serves as a brake that keeps the long isoforms' accelerator effects in check. These findings provide a novel insight into the regulatory roles of NFE2L1 in osteoclastogenesis and highlight that NFE2L1 is essential in regulating bone remodeling and thus may be a valuable therapeutic target for bone disorders.

Project description:RNA helicases are critical regulators of gene expression and therefore strict regulation of their activities is essential to limit promiscuity and ensure target specify. While some RNA helicases appear dedicated for particular substrates others, such as DHX15, are multifunctional, contributing to more than one gene expression process. Protein cofactors play key roles both in modulating RNA helicase activities and also directing them to appropriate substrate RNAs. The largest family of RNA helicase cofactors, with more than 20 members expressed in human cells, are the G-patch proteins, characterised by a glycine-rich domain via which they associate with specific DEAH/RHA helicases. However, the cognate RNA helicases and cellular functions of numerous human G-patch proteins still remain elusive. Here, we reveal that the G-patch protein GPATCH4 is a cofactor of DHX15 that stimulates its ATPase activity. We show that GPATCH4 interacts with DHX15 in the nucleolus via residues in its G-patch domain and co-migrates with pre-ribosomal particles, wherein loss of DHX15 reduces pre-ribosomal co-migration of GPATCH4. Using a combination of high-throughput sequencing approaches (ChIP, CRAC and small RNA-seq), we discovered that GPATCH4 crosslinks to the ribosomal DNA locus and precursor ribosomal RNAs as well as binding to small nucleolar- and small Cajal body-associated- RNAs that guide rRNA and snRNA modifications. In line with this, using RiboMeth-seq analysis to globally analyse rRNA and snRNA 2’-O-methylation revealed that knockout of GPATCH4 impairs methylation at various sites. Utilizing targeted 2’-O-methylation-sensitive RNase H-based cleavage assays, we demonstrated that the regulation of 2’-O-methylation by GPATCH4 is both dependent on, and independent of, its interaction with DHX15. Intriguingly, the ATPase activity of DHX15 is necessary for efficient methylation of DHX15-dependent sites, suggesting a function of DHX15 in regulating snoRNA-guided 2’-O-methylation of rRNA that requires activation by GPATCH4. Overall, our findings not only extend knowledge on RNA helicase regulation by G-patch proteins but also provide important new insights into how the installation of rRNA and snRNA modifications, which are essential for ribosome assembly/function and pre-mRNA splicing, is regulated.

Dataset Information

KAP1 Regulates Regulatory T Cell Function and Proliferation in Both Foxp3-Dependent and -Independent Manners.

Publications

KAP1 Regulates Regulatory T Cell Function and Proliferation in Both Foxp3-Dependent and -Independent Manners.

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