Lifelong persistence of nuclear RNAs in the mouse brain.

Genomic DNA that resides in the nuclei of mammalian neurons can be as old as the organism itself. The life span of nuclear RNAs, which are critical for proper chromatin architecture and transcription regulation, has not been determined in adult tissues. In this work, we identified and characterized nuclear RNAs that do not turn over for at least 2 years in a subset of postnatally born cells in the mouse brain. These long-lived RNAs were stably retained in nuclei in a neural cell type-specific manner and were required for the maintenance of heterochromatin. Thus, the life span of neural cells may depend on both the molecular longevity of DNA for the storage of genetic information and also the extreme stability of RNA for the functional organization of chromatin.

Tipifarnib Potentiates the Antitumor Effects of PI3Kα Inhibition in PIK3CA- and HRAS-Dysregulated HNSCC via Convergent Inhibition of mTOR Activity.

Outcomes for patients with recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) are poor, with median overall survival (OS) ranging from 6 to 18 months. For those who progress on standard-of-care (chemo)immunotherapy, treatment options are limited, necessitating the development of rational therapeutic strategies. Toward this end, we targeted the key HNSCC drivers PI3K-mTOR and HRAS via the combination of tipifarnib, a farnesyltransferase (FTase) inhibitor, and alpelisib, a PI3Kα inhibitor, in multiple molecularly defined subsets of HNSCC. Tipifarnib synergized with alpelisib at the level of mTOR in PI3Kα- or HRAS-dependent HNSCCs, leading to marked cytotoxicity in vitro and tumor regression in vivo. On the basis of these findings, the KURRENT-HN trial was launched to evaluate the effectiveness of this combination in PIK3CA-mutant/amplified and/or HRAS-overexpressing R/M HNSCC. Preliminary evidence supports the clinical activity of this molecular biomarker-driven combination therapy. Combined alpelisib and tipifarnib has potential to benefit >45% of patients with R/M HNSCC. By blocking feedback reactivation of mTORC1, tipifarnib may prevent adaptive resistance to additional targeted therapies, enhancing their clinical utility. SIGNIFICANCE: The mechanistically designed, biomarker-matched strategy of combining alpelisib and tipifarnib is efficacious in PIK3CA- and HRAS-dysregulated head and neck squamous carcinoma and could improve outcomes for many patients with recurrent, metastatic disease. See related commentary by Lee et al., p. 3162.

Improved Method for Efficient Generation of Functional Neurons from Murine Neural Progenitor Cells.

Neuronal culture was used to investigate neuronal function in physiological and pathological conditions. Despite its inevitability, primary neuronal culture remained a gold standard method that requires laborious preparation, intensive training, and animal resources. To circumvent the shortfalls of primary neuronal preparations and efficiently give rise to functional neurons, we combine a neural stem cell culture method with a direct cell type-conversion approach. The lucidity of this method enables the efficient preparation of functional neurons from mouse neural progenitor cells on demand. We demonstrate that induced neurons (NPC-iNs) by this method make synaptic connections, elicit neuronal activity-dependent cellular responses, and develop functional neuronal networks. This method will provide a concise platform for functional neuronal assessments. This indeed offers a perspective for using these characterized neuronal networks for investigating plasticity mechanisms, drug screening assays, and probing the molecular and biophysical basis of neurodevelopmental and neurodegenerative diseases.

The RNA transport factor PHAX is required for proper histone H2AX expression and DNA damage response.

PHAX (phosphorylated adaptor for RNA export) promotes nuclear export of short transcripts of RNA polymerase II such as spliceosomal U snRNA precursors, as well as intranuclear transport of small nucleolar RNAs (snoRNAs). However, it remains unknown whether PHAX has other critical functions. Here we show that PHAX is required for efficient DNA damage response (DDR) via regulation of phosphorylated histone variant H2AX (γH2AX), a key factor for DDR. Knockdown of PHAX led to a significant reduction of H2AX mRNA levels, through inhibition of both transcription of the H2AX gene and nuclear export of H2AX mRNA, one of the shortest mRNAs in the cell. As a result, PHAX-knockdown cells become more sensitive to DNA damage due to a shortage of γH2AX. These results reveal a novel function of PHAX, which secures efficient DDR and hence genome stability.

Tpr regulates the total number of nuclear pore complexes per cell nucleus.

The total number of nuclear pore complexes (NPCs) per nucleus varies greatly between different cell types and is known to change during cell differentiation and cell transformation. However, the underlying mechanisms that control how many nuclear transport channels are assembled into a given nuclear envelope remain unclear. Here, we report that depletion of the NPC basket protein Tpr, but not Nup153, dramatically increases the total NPC number in various cell types. This negative regulation of Tpr occurs via a phosphorylation cascade of extracellular signal-regulated kinase (ERK), the central kinase of the mitogen-activated protein kinase (MAPK) pathway. Tpr serves as a scaffold for ERK to phosphorylate the nucleoporin (Nup) Nup153, which is critical for early stages of NPC biogenesis. Our results reveal a critical role of the Nup Tpr in coordinating signal transduction pathways during cell proliferation and the dynamic organization of the nucleus.

Nup98 recruits the Wdr82-Set1A/COMPASS complex to promoters to regulate H3K4 trimethylation in hematopoietic progenitor cells.

Recent studies have shown that a subset of nucleoporins (Nups) can detach from the nuclear pore complex and move into the nuclear interior to regulate transcription. One such dynamic Nup, called Nup98, has been implicated in gene activation in healthy cells and has been shown to drive leukemogenesis when mutated in patients with acute myeloid leukemia (AML). Here we show that in hematopoietic cells, Nup98 binds predominantly to transcription start sites to recruit the Wdr82-Set1A/COMPASS (complex of proteins associated with Set1) complex, which is required for deposition of the histone 3 Lys4 trimethyl (H3K4me3)-activating mark. Depletion of Nup98 or Wdr82 abolishes Set1A recruitment to chromatin and subsequently ablates H3K4me3 at adjacent promoters. Furthermore, expression of a Nup98 fusion protein implicated in aggressive AML causes mislocalization of H3K4me3 at abnormal regions and up-regulation of associated genes. Our findings establish a function of Nup98 in hematopoietic gene activation and provide mechanistic insight into which Nup98 leukemic fusion proteins promote AML.

Analysis of RNA transport in Xenopus oocytes and mammalian cells.

In eukaryotes, many RNA species are transcribed, processed in the nucleus, and exported to the cytoplasm, where they are destined to function or to be further matured. Some RNAs are even reimported to the nucleus. In addition, many RNAs are localized at specific nuclear bodies before their export and/or after their nuclear reimport. To understand how RNAs are transported, Xenopus oocytes are extremely useful cells, thanks to their large size. RNA transport can be easily examined by microinjecting radioactively or fluorescently labeled RNAs into Xenopus oocytes. Mammalian cultured cells are sometimes useful by virtue of RNA-FISH technique. Here, we describe methods to analyze RNA localization and export using these cells.

p54nrb/NonO and PSF promote U snRNA nuclear export by accelerating its export complex assembly.

The assembly of spliceosomal U snRNPs in metazoans requires nuclear export of U snRNA precursors. Four factors, nuclear cap-binding complex (CBC), phosphorylated adaptor for RNA export (PHAX), the export receptor CRM1 and RanGTP, gather at the m(7)G-cap-proximal region and form the U snRNA export complex. Here we show that the multifunctional RNA-binding proteins p54nrb/NonO and PSF are U snRNA export stimulatory factors. These proteins, likely as a heterodimer, accelerate the recruitment of PHAX, and subsequently CRM1 and Ran onto the RNA substrates in vitro, which mediates efficient U snRNA export in vivo. Our results reveal a new layer of regulation for U snRNA export and, hence, spliceosomal U snRNP biogenesis.

hnRNP C tetramer measures RNA length to classify RNA polymerase II transcripts for export.

Specific RNA recognition is usually achieved by specific RNA sequences and/or structures. However, we show here a mechanism by which RNA polymerase II (Pol II) transcripts are classified according to their length. The heterotetramer of the heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2 measures the length of the transcripts like a molecular ruler, by selectively binding to the unstructured RNA regions longer than 200 to 300 nucleotides. Thus, the tetramer sorts the transcripts into two RNA categories, to be exported as either messenger RNA or uridine-rich small nuclear RNA (U snRNA), depending on whether or not they are longer than the threshold, respectively. Our findings reveal a new function of the C tetramer and highlight the biological importance of RNA recognition by the length.

Multiple factors in the early splicing complex are involved in the nuclear retention of pre-mRNAs in mammalian cells.

Intron-containing pre-mRNAs are retained in the nucleus until they are spliced. This mechanism is essential for proper gene expression. Although the formation of splicing complexes on pre-mRNAs is thought to be responsible for this nuclear retention activity, the details are poorly understood. In mammalian cells, in particular, very little information is available regarding the retention factors. Using a model reporter gene, we show here that U1 snRNP and U2AF but not U2 snRNP are essential for the nuclear retention of pre-mRNAs in mammalian cells, showing that E complex is the major entity responsible for the nuclear retention of pre-mRNAs in mammalian cells. By focusing on factors that bind to the 3'-splice site region, we found that the 65-kD subunit of U2AF (U2AF(65) ) is important for nuclear retention and that its multiple domains have nuclear retention activity per se. We also provide evidence that UAP56, a DExD-box RNA helicase involved in both RNA splicing and export, cooperates with U2AF(65) in exerting nuclear retention activity. Our findings provide new information regarding the pre-mRNA nuclear retention factors in mammalian cells.