Metazoan genes are encrypted with at least two superimposed codes: the genetic code to specify the primary structure of proteins and the splicing code to expand their proteomic output via alternative splicing. Here, we define the specificity of a central regulator of pre-mRNA splicing, the conserved, essential splicing factor SFRS1. Cross-linking immunoprecipitation and high-throughput sequencing (CLIP-seq) identified 23,632 binding sites for SFRS1 in the transcriptome of cultured human embryonic kidney cells. SFRS1 was found to engage many different classes of functionally distinct transcripts including mRNA, miRNA, snoRNAs, ncRNAs, and conserved intergenic transcripts of unknown function. The majority of these diverse transcripts share a purine-rich consensus motif corresponding to the canonical SFRS1 binding site. The consensus site was not only enriched in exons cross-linked to SFRS1 in vivo, but was also enriched in close proximity to splice sites. mRNAs encoding RNA processing factors were significantly overrepresented, suggesting that SFRS1 may broadly influence the post-transcriptional control of gene expression in vivo. Finally, a search for the SFRS1 consensus motif within the Human Gene Mutation Database identified 181 mutations in 82 different genes that disrupt predicted SFRS1 binding sites. This comprehensive analysis substantially expands the known roles of human SR proteins in the regulation of a diverse array of RNA transcripts.[Supplemental material is available online at www.genome.org.]Metazoan genomes are encoded with multiple overlapping layers of information required for the precise control of gene expression. The splicing code has co-evolved with the genetic code and regulates the post-transcriptional expression of protein-coding genes (for review, see Wang and Cooper 2007). In the nucleus, splicing is required to remove intervening sequences (introns) from precursor messenger RNAs (pre-mRNAs) and to correctly join proteinencoding regions (exons) together. Inclusion of an exon into the mature mRNA is regulated by cis-acting RNA elements known as exonic or intronic splicing enhancers and silencers (ESE, ISE, and ESS, ISS, respectively) that function to recruit trans-acting RNAbinding proteins. In the cytoplasm, these same RNA elements are decoded by tRNAs and the ribosome in order to template protein synthesis. Alternative splicing allows a single gene to express many different protein isoforms by including all, some, or none of a specific exon sequence in the mRNA (for review, see Maniatis and Tasic 2002). Current estimates suggest that at least 70% of protein-coding genes undergo alternative splicing (Wang and Cooper 2007). However, understanding how these events are regulated and coordinated represents a major challenge.Classification of functional cis-acting RNA elements on a global scale is required to begin the arduous task of defining the specific outputs from every human gene (for review, see Wang and Burge 2008).

Methylmercury (MeHg) exposure from occupational, environmental, and food sources is a significant threat to public health. MeHg poisonings in adults may result in severe psychological and neurological deficits, and in utero exposures can confer embryonic defects and developmental delays. Recent epidemiological and vertebrate studies suggest that MeHg exposure may also contribute to dopamine (DA) neuron vulnerability and the propensity to develop Parkinson's disease (PD). In this study, we describe a Caenorhabditis elegans model of MeHg toxicity that shows that low, chronic exposure confers embryonic defects, developmental delays, decreases in brood size and animal viability, and DA neuron degeneration. Toxicant exposure results in the robust induction of the glutathione-S-transferases (GSTs) gst-4 and gst-38 that are largely dependent on the PD-associated phase II antioxidant transcription factor SKN-1/Nrf2. We also demonstrate that the expression of SKN-1, a protein previously localized to a small subset of chemosensory neurons and intestinal cells in the nematode, is also expressed in the DA neurons, and a reduction in SKN-1 gene expression increases MeHg-induced animal vulnerability and DA neuron degeneration. These studies recapitulate fundamental hallmarks of MeHg-induced mammalian toxicity, identify a key molecular regulator of toxicant-associated whole-animal and DA neuron vulnerability, and suggest that the nematode will be a useful in vivo tool to identify and characterize mediators of MeHg-induced developmental and DA neuron pathologies.

Ferritin, a 24-mer heteropolymer of heavy (H) and light (L) subunits, is the main cellular iron storage protein and plays a pivotal role in iron homeostasis by modulating free iron levels thus reducing radical-mediated damage. The H subunit has ferroxidase activity (converting Fe(II) to Fe(III)), while the L subunit promotes iron nucleation and increases ferritin stability. Previous studies on the H gene (Fth) in mice have shown that complete inactivation of Fth is lethal during embryonic development, without ability to compensate by the L subunit. In humans, homozygous loss of the L gene (FTL) is associated with generalized seizure and atypical restless leg syndrome, while mutations in FTL cause a form of neurodegeneration with brain iron accumulation. Here we generated mice with genetic ablation of the Fth and Ftl genes. As previously reported, homozygous loss of the Fth allele on a wild-type Ftl background was embryonic lethal, whereas knock-out of the Ftl allele (Ftl-/-) led to a significant decrease in the percentage of Ftl-/- newborn mice. Analysis of Ftl-/- mice revealed systemic and brain iron dyshomeostasis, without any noticeable signs of neurodegeneration. Our findings indicate that expression of the H subunit can rescue the loss of the L subunit and that H ferritin homopolymers have the capacity to sequester iron in vivo. We also observed that a single allele expressing the H subunit is not sufficient for survival when both alleles encoding the L subunit are absent, suggesting the need of some degree of complementation between the subunits as well as a dosage effect.

Exposure to high levels of manganese (Mn) results in a neurological condition termed manganism, which is characterized by oxidative stress, abnormal dopamine (DA) signaling, and cell death. Epidemiological evidence suggests correlations with occupational exposure to Mn and the development of the movement disorder Parkinson's disease (PD), yet the molecular determinants common between the diseases are ill-defined. Glutathione S-transferases (GSTs) of the class pi (GSTπ) are phase II detoxification enzymes that conjugate both endogenous and exogenous compounds to glutathione to reduce cellular oxidative stress, and their decreased expression has recently been implicated in PD progression. In this study we demonstrate that a Caenorhabditis elegans GSTπ homologue, GST-1, inhibits Mn-induced DA neuron degeneration. We show that GST-1 is expressed in DA neurons, Mn induces GST-1 gene and protein expression, and GST-1-mediated neuroprotection is dependent on the PD-associated transcription factor Nrf2/SKN-1, as a reduction in SKN-1 gene expression results in a decrease in GST-1 protein expression and an increase in DA neuronal death. Furthermore, decreases in gene expression of the SKN-1 inhibitor WDR-23 or the GSTTπ-binding cell death activator JNK/JNK-1 result in an increase in resistance to the metal. Finally, we show that the Mn-induced DA neuron degeneration is independent of the dopamine transporter DAT, but is largely dependent on the caspases CED-3 and the novel caspase CSP-1. This study identifies a C. elegans Nrf2/SKN-1-dependent GSTπ homologue, cell death effectors of GSTTπ-associated xenobiotic-induced pathology, and provides the first in vivo evidence that a phase II detoxification enzyme may modulate DA neuron vulnerability in manganism.

Aluminum (Al3+) is the most prevalent metal in the earth's crust, and is a known human neurotoxicant. Al3+ has been shown to accumulate in the substantia nigra of Parkinson's disease (PD) patients, and epidemiological studies suggest correlations between Al3+ exposure and the propensity to develop both PD and the amyloid plaque-associated disorder Alzheimer's disease (AD). Although Al3+ exposures have been associated with the development of the most common neurodegenerative disorders, the molecular mechanism involved in Al3+ transport in neurons and subsequent cellular death has remained elusive. In this study we show that a brief exposure to Al3+ decreases mitochondrial membrane potential and cellular ATP levels, and confers dopamine (DA) neuron degeneration in the genetically tractable nematode Caenorhabditis elegans (C. elegans). Al3+ exposure also exacerbates DA neuronal death conferred by the human PD-associated protein α-synuclein. DA neurodegeneration is dependent on SMF-3, a homologue to the human divalent metal transporter (DMT-1), as a functional null mutation partially inhibits the cell death. We also show that SMF-3 is expressed in DA neurons, Al3+ exposure results in a significant decrease in protein levels, and the neurodegeneration is partially dependent on the PD-associated transcription factor Nrf2/SKN-1 and caspase Apaf1/CED-4. Furthermore we provide evidence that the deletion of SMF-3 confers Al3+-resistance due to sequestration of Al3+ into an intracellular compartment. This study describes a novel model for Al3+-induced DA neurodegeneration and provides the first molecular evidence of an animal Al3+ transporter.