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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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.