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Essential nucleotides direct neuron-specific splicing of gamma 2 pre-mRNA.
RNA. 1996 Jul; 2(7):682-98.RNA

Abstract

Tissue-and stage-specific pre-mRNA splicing events are prevalent in mammals, yet molecular details are lacking about these important mechanisms of posttranscriptional gene control. In this study, we investigate the regulated splicing of rat gamma 2 pre-mRNA, a subunit of the GABAA receptor, as a step toward understanding the molecular basis of a neuron-specific splicing event involving cassette exon selection. Cell-and substrate-specific regulation of gamma 2 pre-mRNA is recapitulated in a neuronal cell line derived from the cerebellum, which produces enhanced levels of the exon-selected mRNA. In contrast, a control cell line derived from non-neuronal cells of the pituitary produces prominent levels of the unregulated, exon-skipped mRNA. The cerebellar and pituitary cell lines are well matched in overall splicing efficiency and produce an invariant pattern of splicing for a control substrate, which is alternatively spliced but not regulated in this system. The appropriateness of the two cell lines is indicated by an extended mRNA mapping experiment, which documents the region-specific switch in exon selection throughout rat brain. Using this pair of cell lines, we show that large intron segments flanking the regulated exon are dispensable for regulation. These intron regions have been deleted to generate a minimal splicing substrate for the purpose of identifying essential RNA elements. In this context, we show that essential nucleotides are located at positions +7, +8, and +9 of the regulated exon and in a 9-nt adenosine-rich region of the adjacent 3' splice site. Due to the proximity and base complementarity of the required nucleotides, experiments were devised to test models involving the recognition of two single-stranded signals, or one duplex RNA signal. These results clearly disfavor the duplex RNA recognition model and indicate that the required regions are recognized as independent, single strands in neuronal cells. A weak 5' splice site adjacent to the regulated exon is required as a third essential element. Although the importance of a weak 5' splice site is common to other regulated systems such as NCAM, the essential nucleotides in the exon and 3' splice site region defined in this study for gamma 2 splicing regulation are novel.

Authors+Show Affiliations

Howard Hughes Medical Institute, University of Pittsburgh, Pennsylvania 15260, USA.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8756411

Citation

Zhang, L, et al. "Essential Nucleotides Direct Neuron-specific Splicing of Gamma 2 Pre-mRNA." RNA (New York, N.Y.), vol. 2, no. 7, 1996, pp. 682-98.
Zhang L, Ashiya M, Sherman TG, et al. Essential nucleotides direct neuron-specific splicing of gamma 2 pre-mRNA. RNA. 1996;2(7):682-98.
Zhang, L., Ashiya, M., Sherman, T. G., & Grabowski, P. J. (1996). Essential nucleotides direct neuron-specific splicing of gamma 2 pre-mRNA. RNA (New York, N.Y.), 2(7), 682-98.
Zhang L, et al. Essential Nucleotides Direct Neuron-specific Splicing of Gamma 2 Pre-mRNA. RNA. 1996;2(7):682-98. PubMed PMID: 8756411.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Essential nucleotides direct neuron-specific splicing of gamma 2 pre-mRNA. AU - Zhang,L, AU - Ashiya,M, AU - Sherman,T G, AU - Grabowski,P J, PY - 1996/7/1/pubmed PY - 1996/7/1/medline PY - 1996/7/1/entrez SP - 682 EP - 98 JF - RNA (New York, N.Y.) JO - RNA VL - 2 IS - 7 N2 - Tissue-and stage-specific pre-mRNA splicing events are prevalent in mammals, yet molecular details are lacking about these important mechanisms of posttranscriptional gene control. In this study, we investigate the regulated splicing of rat gamma 2 pre-mRNA, a subunit of the GABAA receptor, as a step toward understanding the molecular basis of a neuron-specific splicing event involving cassette exon selection. Cell-and substrate-specific regulation of gamma 2 pre-mRNA is recapitulated in a neuronal cell line derived from the cerebellum, which produces enhanced levels of the exon-selected mRNA. In contrast, a control cell line derived from non-neuronal cells of the pituitary produces prominent levels of the unregulated, exon-skipped mRNA. The cerebellar and pituitary cell lines are well matched in overall splicing efficiency and produce an invariant pattern of splicing for a control substrate, which is alternatively spliced but not regulated in this system. The appropriateness of the two cell lines is indicated by an extended mRNA mapping experiment, which documents the region-specific switch in exon selection throughout rat brain. Using this pair of cell lines, we show that large intron segments flanking the regulated exon are dispensable for regulation. These intron regions have been deleted to generate a minimal splicing substrate for the purpose of identifying essential RNA elements. In this context, we show that essential nucleotides are located at positions +7, +8, and +9 of the regulated exon and in a 9-nt adenosine-rich region of the adjacent 3' splice site. Due to the proximity and base complementarity of the required nucleotides, experiments were devised to test models involving the recognition of two single-stranded signals, or one duplex RNA signal. These results clearly disfavor the duplex RNA recognition model and indicate that the required regions are recognized as independent, single strands in neuronal cells. A weak 5' splice site adjacent to the regulated exon is required as a third essential element. Although the importance of a weak 5' splice site is common to other regulated systems such as NCAM, the essential nucleotides in the exon and 3' splice site region defined in this study for gamma 2 splicing regulation are novel. SN - 1355-8382 UR - https://www.unboundmedicine.com/medline/citation/8756411/Essential_nucleotides_direct_neuron_specific_splicing_of_gamma_2_pre_mRNA_ L2 - http://www.rnajournal.org/cgi/pmidlookup?view=long&pmid=8756411 DB - PRIME DP - Unbound Medicine ER -