Biomedical subjects

Paula J Grabowski

Publications and source records attributed to Paula J Grabowski.

6 recordsLinked to original sources

PubMed2005

Evidence for direct roles of two additional factors, SECp43 and soluble liver antigen, in the selenoprotein synthesis machinery.

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA([Ser]Sec), but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2'-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA([Ser]Sec), and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA([Ser]Sec) and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA([Ser]Sec). SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA([Ser]Sec) between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA([Ser]Sec) in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA([Ser]Sec) and the intracellular location of SLA.

Animals↗

PubMed2005

A combinatorial code for splicing silencing: UAGG and GGGG motifs.

Alternative pre-mRNA splicing is widely used to regulate gene expression by tuning the levels of tissue-specific mRNA isoforms. Few regulatory mechanisms are understood at the level of combinatorial control despite numerous sequences, distinct from splice sites, that have been shown to play roles in splicing enhancement or silencing. Here we use molecular approaches to identify a ternary combination of exonic UAGG and 5'-splice-site-proximal GGGG motifs that functions cooperatively to silence the brain-region-specific CI cassette exon (exon 19) of the glutamate NMDA R1 receptor (GRIN1) transcript. Disruption of three components of the motif pattern converted the CI cassette into a constitutive exon, while predominant skipping was conferred when the same components were introduced, de novo, into a heterologous constitutive exon. Predominant exon silencing was directed by the motif pattern in the presence of six competing exonic splicing enhancers, and this effect was retained after systematically repositioning the two exonic UAGGs within the CI cassette. In this system, hnRNP A1 was shown to mediate silencing while hnRNP H antagonized silencing. Genome-wide computational analysis combined with RT-PCR testing showed that a class of skipped human and mouse exons can be identified by searches that preserve the sequence and spatial configuration of the UAGG and GGGG motifs. This analysis suggests that the multi-component silencing code may play an important role in the tissue-specific regulation of the CI cassette exon, and that it may serve more generally as a molecular language to allow for intricate adjustments and the coordination of splicing patterns from different genes.

Alternative Splicing↗

PubMed2005

Splicing-active nuclear extracts from rat brain.

In the nervous system, alternative pre-mRNA splicing generates the diverse protein machineries needed for cell excitation and synaptic communication. Yet, many questions remain about how these mechanisms are regulated by RNA binding proteins in the environment of differentiated cells and tissues. Here, we describe the preparation and use of splicing active nuclear extracts derived from the cerebellum and cerebral cortex regions of rat brain as a resource for in vitro studies. These tissue-specific extracts promote the neuron-specific pathway of splicing, and display characteristic changes in hnRNP protein function and expression. These extracts can be used in combination with affinity selection and depletion/complementation assays to identify regulatory factors and to characterize their interactions and effects on spliceosome assembly. The ability to prepare extracts from brain regions at a range of postnatal ages provides opportunities to address related questions as a function of cell differentiation. These neuronal extracts may also be valuable for the development of in vitro assays to elucidate other neuron-specific RNA processing pathways, such as 3' end formation, RNA editing, or miRNA maturation.

Animals↗

PubMed2002

Function of quaking in myelination: regulation of alternative splicing.

Proteomic diversity is frequently achieved by alternative RNA-splicing events that can be fine-tuned in tissue-specific and developmentally regulated ways. Understanding this type of genetic regulation is compelling because of the extensive complexity of alternative splicing found in the nervous system. quaking (qk), one of the classical mouse dysmyelination mutants, is defective for the expression of myelin-associated glycoprotein (MAG), and the misregulation of MAG pre-mRNA alternative splicing is implicated as a causal factor. The qk locus encodes several RNA-binding proteins with heterogeneous nuclear ribonucleoprotein K-type homology, a characteristic of several known alternative splicing regulators. Here we test the nuclear-localized qk isoform (QKI-5) for its ability to regulate alternative splicing of MAG pre-mRNA in transient coexpression assays. QKI-5 exhibits properties of a negative regulator of MAG exon 12 alternative splicing. An intronic sequence element required for the repressive function and binding of QKI-5 is also identified. Direct evidence for irregularities in alternative splicing of MAG and other myelin protein transcripts in the qk mouse is demonstrated.

Alternative Splicing↗

PubMed2002

Mutations in RRM4 uncouple the splicing repression and RNA-binding activities of polypyrimidine tract binding protein.

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequence-specific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays.

Alternative Splicing↗

PubMed2002

Region-specific alternative splicing in the nervous system: implications for regulation by the RNA-binding protein NAPOR.

Alternative RNA splicing generates extensive proteomic diversity in the nervous system, yet few neural-specific RNA binding proteins have been implicated in splicing control. Here we show that the biochemical properties and spatial expression of mouse neuroblastoma apoptosis-related RNA-binding protein (NAPOR; also called NAPOR-1) are consistent with its roles in the regulation of the exon 5 and exon 21 splicing events of the N-methyl-D-aspartate (NMDA) receptor R1 transcript. NAPOR, which is closely related to CUG binding protein 2 (CUG-BP2), promotes exon 21 and represses exon 5 splicing in functional coexpression assays. These NMDA mRNA isoforms are distributed, in vivo, in a region-specific manner in rat brain, such that high levels of exon 21 selection and exon 5 skipping coincide with high NAPOR mRNA expression in the forebrain. Within the forebrain, this spatial correspondence is most striking in the visual cortex. In contrast, low NAPOR expression coincides with the reciprocal pattern of alternative splicing in the hindbrain. Complementary experiments demonstrate a tissue-specific distribution of NAPOR, CUG-BP, and other highly related proteins within the nervous system as assayed by probing forebrain and hindbrain nuclear extracts with monoclonal antibody, mAb 3B1. Thus, NAPOR may be one of a group of closely related proteins involved in splicing regulation within the brain. An intronic RNA element responsible for the silencing of exon 21 splicing is identified by mutational analysis and shown to bind directly to recombinant NAPOR protein, suggesting a model in which exon 21 selection is positively regulated by an antirepression mechanism of action.

Alternative Splicing↗