Hybridization-proximity labeling reveals spatially ordered interactions of nuclear RNA compartments.

The ability of RNAs to form specific contacts with other macromolecules provides an important mechanism for subcellular compartmentalization. Here we describe a suite of hybridization-proximity (HyPro) labeling technologies for unbiased discovery of proteins (HyPro-MS) and transcripts (HyPro-seq) associated with RNAs of interest in genetically unperturbed cells. As a proof of principle, we show that HyPro-MS and HyPro-seq can identify both known and previously unexplored spatial neighbors of the noncoding RNAs 45S, NEAT1, and PNCTR expressed at markedly different levels. Notably, HyPro-seq uncovers an extensive repertoire of incompletely processed, adenosine-to-inosine-edited transcripts accumulating at the interface between their encoding chromosomal regions and the NEAT1-containing paraspeckle compartment. At least some of these targets require NEAT1 for their optimal expression. Overall, this study provides a versatile toolkit for dissecting RNA interactomes in diverse biomedical contexts and expands our understanding of the functional architecture of the mammalian nucleus.

A Short Tandem Repeat-Enriched RNA Assembles a Nuclear Compartment to Control Alternative Splicing and Promote Cell Survival.

Functions of many long noncoding RNAs (lncRNAs) depend on their ability to interact with multiple copies of specific RNA-binding proteins (RBPs). Here, we devised a workflow combining bioinformatics and experimental validation steps to systematically identify RNAs capable of multivalent RBP recruitment. This uncovered a number of previously unknown transcripts encoding high-density RBP recognition arrays within genetically normal short tandem repeats. We show that a top-scoring hit in this screen, lncRNA PNCTR, contains hundreds of pyrimidine tract-binding protein (PTBP1)-specific motifs allowing it to sequester a substantial fraction of PTBP1 in a nuclear body called perinucleolar compartment. Importantly, PNCTR is markedly overexpressed in a variety of cancer cells and its downregulation is sufficient to induce programmed cell death at least in part by stimulating PTBP1 splicing regulation activity. This work expands our understanding of the repeat-containing fraction of the human genome and illuminates a novel mechanism driving malignant transformation of cancer cells.

Alternative exon definition events control the choice between nuclear retention and cytoplasmic export of U11/U12-65K mRNA.

Cellular homeostasis of the minor spliceosome is regulated by a negative feed-back loop that targets U11-48K and U11/U12-65K mRNAs encoding essential components of the U12-type intron-specific U11/U12 di-snRNP. This involves interaction of the U11 snRNP with an evolutionarily conserved splicing enhancer giving rise to unproductive mRNA isoforms. In the case of U11/U12-65K, this mechanism controls the length of the 3' untranslated region (3'UTR). We show that this process is dynamically regulated in developing neurons and some other cell types, and involves a binary switch between translation-competent mRNAs with a short 3'UTR to non-productive isoforms with a long 3'UTR that are retained in the nucleus or/and spliced to the downstream amylase locus. Importantly, the choice between these alternatives is determined by alternative terminal exon definition events regulated by conserved U12- and U2-type 5' splice sites as well as sequence signals used for pre-mRNA cleavage and polyadenylation. We additionally show that U11 snRNP binding to the U11/U12-65K mRNA species with a long 3'UTR is required for their nuclear retention. Together, our studies uncover an intricate molecular circuitry regulating the abundance of a key spliceosomal protein and shed new light on the mechanisms limiting the export of non-productively spliced mRNAs from the nucleus to the cytoplasm.

Coordinated regulation of neuronal mRNA steady-state levels through developmentally controlled intron retention.

Differentiated cells acquire unique structural and functional traits through coordinated expression of lineage-specific genes. An extensive battery of genes encoding components of the synaptic transmission machinery and specialized cytoskeletal proteins is activated during neurogenesis, but the underlying regulation is not well understood. Here we show that genes encoding critical presynaptic proteins are transcribed at a detectable level in both neurons and nonneuronal cells. However, in nonneuronal cells, the splicing of 3'-terminal introns within these genes is repressed by the polypyrimidine tract-binding protein (Ptbp1). This inhibits the export of incompletely spliced mRNAs to the cytoplasm and triggers their nuclear degradation. Clearance of these intron-containing transcripts occurs independently of the nonsense-mediated decay (NMD) pathway but requires components of the nuclear RNA surveillance machinery, including the nuclear pore-associated protein Tpr and the exosome complex. When Ptbp1 expression decreases during neuronal differentiation, the regulated introns are spliced out, thus allowing the accumulation of translation-competent mRNAs in the cytoplasm. We propose that this mechanism counters ectopic and precocious expression of functionally linked neuron-specific genes and ensures their coherent activation in the appropriate developmental context.

Functional impact of splice isoform diversity in individual cells.

Alternative pre-mRNA splicing provides an effective means for expanding coding capacity of eukaryotic genomes. Recent studies suggest that co-expression of different splice isoforms may increase diversity of RNAs and proteins at a single-cell level. A pertinent question in the field is whether such co-expression is biologically meaningful or, rather, represents insufficiently stringent splicing regulation. Here we argue that isoform co-expression may produce functional outcomes that are difficult and sometimes impossible to achieve using other regulation strategies. Far from being a 'splicing noise', co-expression is often established through co-ordinated activity of specific cis-elements and trans-acting factors. Further work in this area may uncover new biological functions of alternative splicing (AS) and generate important insights into mechanisms allowing different cell types to attain their unique molecular identities.

MicroRNA miR-124 controls the choice between neuronal and astrocyte differentiation by fine-tuning Ezh2 expression.

Polycomb group protein Ezh2 is a histone H3 Lys-27 histone methyltransferase orchestrating an extensive epigenetic regulatory program. Several nervous system-specific genes are known to be repressed by Ezh2 in stem cells and derepressed during neuronal differentiation. However, the molecular mechanisms underlying this regulation remain poorly understood. Here we show that Ezh2 levels are dampened during neuronal differentiation by brain-enriched microRNA miR-124. Expression of miR-124 in a neuroblastoma cells line was sufficient to up-regulate a significant fraction of nervous system-specific Ezh2 target genes. On the other hand, naturally elevated expression of miR-124 in embryonic carcinoma cells undergoing neuronal differentiation correlated with down-regulation of Ezh2 levels. Importantly, overexpression of Ezh2 mRNA with a 3'-untranslated region (3'-UTR) lacking a functional miR-124 binding site, but not with the wild-type Ezh2 3'-UTR, hampered neuronal and promoted astrocyte-specific differentiation in P19 and embryonic mouse neural stem cells. Overall, our results uncover a molecular mechanism that allows miR-124 to balance the choice between alternative differentiation possibilities through fine-tuning the expression of a critical epigenetic regulator.

Regulation of gene expression in mammalian nervous system through alternative pre-mRNA splicing coupled with RNA quality control mechanisms.

Eukaryotic gene expression is orchestrated on a genome-wide scale through several post-transcriptional mechanisms. Of these, alternative pre-mRNA splicing expands the proteome diversity and modulates mRNA stability through downstream RNA quality control (QC) pathways including nonsense-mediated decay (NMD) of mRNAs containing premature termination codons and nuclear retention and elimination (NRE) of intron-containing transcripts. Although originally identified as mechanisms for eliminating aberrant transcripts, a growing body of evidence suggests that NMD and NRE coupled with deliberate changes in pre-mRNA splicing patterns are also used in a number of biological contexts for deterministic control of gene expression. Here we review recent studies elucidating molecular mechanisms and biological significance of these gene regulation strategies with a specific focus on their roles in nervous system development and physiology. This article is part of a Special Issue entitled 'RNA and splicing regulation in neurodegeneration'.

Streamlined platform for short hairpin RNA interference and transgenesis in cultured mammalian cells.

Sequence-specific gene silencing by short hairpin (sh) RNAs has recently emerged as an indispensable tool for understanding gene function and a promising avenue for drug discovery. However, a wider biomedical use of this approach is hindered by the lack of straightforward methods for achieving uniform expression of shRNAs in mammalian cell cultures. Here we report a high-efficiency and low-background (HILO) recombination-mediated cassette exchange (RMCE) technology that yields virtually homogeneous cell pools containing doxycycline-inducible shRNA elements in a matter of days and with minimal efforts. To ensure immediate utility of this approach for a wider research community, we modified 11 commonly used human (A549, HT1080, HEK293T, HeLa, HeLa-S3, and U2OS) and mouse (CAD, L929, N2a, NIH 3T3, and P19) cell lines to be compatible with the HILO-RMCE process. Because of its technical simplicity and cost efficiency, the technology will be advantageous for both low- and high-throughput shRNA experiments. We also provide evidence that HILO-RMCE will facilitate a wider range of molecular and cell biology applications by allowing one to rapidly engineer cell populations expressing essentially any transgene of interest.

Alternative splicing coupled to nonsense-mediated decay coordinates downregulation of non-neuronal genes in developing mouse neurons.

BACKGROUND: The functional coupling between alternative pre-mRNA splicing (AS) and the mRNA quality control mechanism called nonsense-mediated decay (NMD) can modulate transcript abundance. Previous studies have identified several examples of such a regulation in developing neurons. However, the systems-level effects of AS-NMD in this context are poorly understood. RESULTS: We developed an R package, factR2, which offers a comprehensive suite of AS-NMD analysis functions. Using this tool, we conducted a longitudinal analysis of gene expression in pluripotent stem cells undergoing induced neuronal differentiation. Our analysis uncovers hundreds of AS-NMD events with significant potential to regulate gene expression. Notably, this regulation is significantly overrepresented in specific functional groups of developmentally downregulated genes. Particularly strong association with gene downregulation is detected for alternative cassette exons stimulating NMD upon their inclusion into mature mRNA. By combining bioinformatic analyses with CRISPR/Cas9 genome editing and other experimental approaches we show that NMD-stimulating cassette exons regulated by the RNA-binding protein PTBP1 dampen the expression of their genes in developing neurons. We also provided evidence that the inclusion of NMD-stimulating cassette exons into mature mRNAs is temporally coordinated with NMD-independent gene repression mechanisms. CONCLUSIONS: Our study provides an accessible workflow for the discovery and prioritization of AS-NMD targets. It further argues that the AS-NMD pathway plays a widespread role in developing neurons by facilitating the downregulation of functionally related non-neuronal genes.

Analysis of RNA-containing compartments by hybridization and proximity labeling in cultured human cells.

This protocol describes a hybridization-proximity labeling (HyPro) approach for identification of proteins and RNAs co-localizing with a transcript of interest in genetically unperturbed cells. It outlines steps required for purification of a recombinant HyPro enzyme, hybridization of fixed and permeabilized cells with digoxigenin-labeled probes, HyPro enzyme binding, proximity biotinylation, and downstream analyses of the biotinylated products. Although the protocol is optimized for relatively abundant noncoding transcripts, recommendations are provided for improving the signal-to-noise ratio in case of scarcer RNA "baits." For complete details on the use and execution of this protocol, please refer to Yap et al. (2021).

Clinically reported heterozygous mutations in the PINK1 kinase domain exert a gene dosage effect.

Mutations in the gene encoding phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) have been associated with the loss of dopaminergic neurons characteristic of familial and sporadic Parkinson disease. We developed an in vitro system of stable human dopaminergic neuronal cell lines coexpressing an equivalent copy of normal and mutant PINK1 to simulate "heterozygous" and "homozygous" states in patients. Mutants in the N-terminus, C-terminus, and kinase domain were generated and cloned into a two-gene mammalian expression vector to generate stable mammalian expression cell lines producing an equivalent copy number of wild-type/mutant PINK1. The cell lines were subjected to oxidative stress and the rate of apoptosis and change in mitochondrial membrane potential (DeltaPsi(m)) were assessed. Cell lines expressing kinase and C-terminus mutants exhibited a greater rate of apoptosis and decrease in DeltaPsi(m), and increased time-dependent cell loss when subjected to oxidative stress compared to the wild-type. Cell lines expressing two copies of kinase mutants exhibited a greater apoptosis rate and DeltaPsi(m) decrease than those expressing one copy of the mutant. In time-dependent experiments, there was a significant difference between "homozygous," "heterozygous," and wild-type cell lines, with decreasing cell survival in cell lines expressing mutant copies of PINK1 compared to the wild-type. We provided the first experimental evidence that clinically reported PINK1 heterozygous mutations exert a gene dosage effect, suggesting that haploinsufficiency of PINK1 is the most likely mechanism that increased the susceptibility to dopaminergic cellular loss.

Multiparity and the risk of premenopausal breast cancer: different effects across ethnic groups in Singapore.

BACKGROUND: The relationship between multiparity and premenopausal breast cancer risk is different in Caucasian, African-American and Hispanic women. For Asian women, this relationship has never been well studied. METHODS: Within the Singapore Birth Registry, we selected all women who had a first child between 1986 and 2002 (169,936 Chinese, 40,521 Malay, 17,966 Indian). We linked them to the Singapore Cancer Registry data to identify those who developed breast cancer after childbirth (n = 527). We used multivariate Cox analysis to examine the relationship between parity, ethnicity and premenopausal breast cancer risk. RESULTS: Compared to Chinese, Malay women had increased and Indian women had decreased risks of premenopausal breast cancer (adjusted Hazard Ratios [HRadj] 1.25 [1.0-1.6] and 0.48 [0.3-0.8] respectively). Multiparity did not modify the risk of premenopausal breast cancer in Chinese and Indians. In Malays there was a significant risk reduction with increasing parity (P (trend )0.037). Malay women with one, two and >or=3 children had premenopausal breast cancer risks (HR(adj)) of 1.86 (1.2-3.0), 1.52 (1.1-2.2) and 0.87 (0.6-1.3) respectively compared to their Chinese counterparts. CONCLUSIONS: The impact of multiparity on premenopausal breast cancer risk differs across ethnic groups in Singapore. Increasing parity reduces the risk of premenopausal breast cancer in Malay, but not in Chinese and Indian women. Uniparous Malay women have twice the risk of premenopausal breast cancer compared to uniparous Chinese. This excess risk disappears after giving birth to >or=3 children. Indian women have lower premenopausal breast cancer risks than Chinese, regardless of their parity status.

Polarizing the Neuron through Sustained Co-expression of Alternatively Spliced Isoforms.

Alternative splicing (AS) is an important source of proteome diversity in eukaryotes. However, how this affects protein repertoires at a single-cell level remains an open question. Here, we show that many 3'-terminal exons are persistently co-expressed with their alternatives in mammalian neurons. In an important example of this scenario, cell polarity gene Cdc42, a combination of polypyrimidine tract-binding, protein-dependent, and constitutive splicing mechanisms ensures a halfway switch from the general (E7) to the neuron-specific (E6) alternative 3'-terminal exon during neuronal differentiation. Perturbing the nearly equimolar E6/E7 ratio in neurons results in defects in both axonal and dendritic compartments and suggests that Cdc42E7 is involved in axonogenesis, whereas Cdc42E6 is required for normal development of dendritic spines. Thus, co-expression of a precise blend of functionally distinct splice isoforms rather than a complete switch from one isoform to another underlies proper structural and functional polarization of neurons.

Deep vein thrombosis and malignancy: a surgical oncologist's perspective.

Oncology patients are at increased risk of developing deep vein thrombosis (DVT) and its potentially fatal sequel, pulmonary embolism. This is due to multiple factors, including the presence of the malignancy itself, comorbid factors and therapy-related interventions. Issues that are peculiar to venous thrombosis in the oncology setting are discussed, based on a MEDLINE search of the English literature. These include the need to screen for malignancy in idiopathic DVT, a high index of suspicion for venous thrombosis in the cancer patient, the use of vena cava filters, and the anti-neoplastic effects of heparin. Asian patients appear to have a lower incidence of DVT compared to Caucasians. A recommended regimen for prophylaxis of DVT must take into account the varying thrombosis risk associated with different malignancies. Cancer patients not undergoing abdominal, pelvic or orthopaedic surgery (e.g. mastectomy) should use elastic compression stockings and be mobilized early, whereas low-molecular-weight heparin should be given to those undergoing more major surgery. In advanced malignancy, treatment of DVT palliates symptoms. These patients may need long-term anticoagulation with warfarin.

Factors influencing arm and axillary symptoms after treatment for node negative breast carcinoma.

BACKGROUND: The purpose of the current study was to identify the factors that contribute to postoperative arm symptoms following breast conserving surgery in a well-defined cohort of node negative breast carcinoma patients. METHODS: A convenience sample of 370 women >/= 50 years of age with node negative breast carcinoma who were participants in a randomized controlled trial designed to assess the need for breast radiation in addition to tamoxifen were surveyed. Axillary dissection was optional for patients 65 years or older and who were clinically node negative. RESULTS: A total of 65.1% (241/370) of women had ipsilateral shoulder or arm symptoms. Multivariate analysis revealed that axillary dissection, breast radiation, and younger age (odds ratio = 11.2, 1.65, and 3.8 respectively) were significantly (P < 0.05) associated with increased ipsilateral shoulder or arm symptoms. Treatment with axillary dissection and breast radiation were significant factors (P < 0.02) associated with the self-reporting of arm swelling (odds ratio = 4.4 and 2.0, respectively). Patients 70 years old or greater reported significantly fewer arm symptoms (odds ratio = 0.26, P < 0.05) after axillary dissection. CONCLUSIONS: Arm symptoms were present in about 80% of patients who underwent breast conserving surgery, axillary dissection, and breast radiation in the current study. These symptoms were significantly associated with the use of axillary dissection, breast radiation, and younger age. Older patients experienced fewer arm symptoms after standard treatment for node negative breast carcinoma, and thus older age should not be a contraindication to axillary dissection.

Use of alternative therapy in postmenopausal breast cancer patients treated with tamoxifen after surgery.

The goal of this study was to determine the frequency of alternative therapy use in postmenopausal women with early stage breast cancer who were enrolled in a randomized clinical trial designed to determine the value of breast irradiation after treatment with breast-conserving surgery and tamoxifen. A questionnaire was given to 300 patients, ages 52 to 90 years, after completion of radiation therapy (if any). Of the 290 respondents, 78 (27%) had used some form of alternative therapy. Of these, 60.3% started after the diagnosis of breast cancer. Users of alternative therapies were significantly younger than nonusers (67.0 +/- 8.4 years versus 70.0 +/- 8.7 years, p = 0.009) and they used a median of one type of therapy per person (range 1-13). Users of alternative therapies were more likely to have experienced symptoms (stiffness, pain, numbness, or swelling) in the ipsilateral shoulder or arm after treatment of their breast cancers compared to nonusers (odds ratio [OR] = 2.0, p = 0.02). This relationship between alternative therapy use and symptoms was strongest in the group who started alternative therapies after breast cancer diagnosis (OR = 2.1, p = 0.05). On multivariate analysis, younger age and radiotherapy treatment were related to alternative therapy use. In conclusion, 27% of patients with early stage breast cancer used alternative therapy. Users were more likely to be younger and to experience shoulder or arm symptoms after breast-conserving surgery with radiation.

2-Octylcynanoacrylate-assisted microvascular anastomosis in a rat model: long-term biomechanical properties and histological changes.

The aim of this study was to establish the long-term biomechanical and histological properties of 2-octylcyanoacrylate-assisted microvascular anastomosis over conventional suture-only anastomosis in the laboratory rat model. The biomechanical and histological properties of three groups of vessels were compared: 1) vessels with 2-octylcyanoacrylate-assisted anastomoses (study group); 2) vessels with suture-only anastomoses (control group); and 3) normal unoperated vessels (sham group). In total, 144 adult rats were used, and these were studied at 1 week, 1 month, 3 months, and 6 months postanastomosis. At 6 months, the tensile strength of study vessels was significantly higher than control vessels. The stiffness of study and control vessels was similar at all time intervals. Histologically, there was no evidence that 2- octylcyanoacrylate caused toxicity to vessel walls, and there was less perivasacular foreign-body giant-cell reaction in the study group compared to the control group. Long-term follow-up showed that microvascular anastomosis with 2-octylcyanoacrylate in rat femoral arteries had superior tensile strength and similar stiffness to vessels anastomosed with sutures only, without adverse effects to surrounding tissues.