Journal: Nucleic Acids Research
Article Title: The human branchpoint-interacting stem-loop sequence and structure regulates U2 snRNA expression, branchpoint recognition, and the transcriptome
doi: 10.1093/nar/gkag245
Figure Lengend Snippet: Reproducing an orthogonal splicing system for U2 snRNA BSL structure/function studies in human cells. ( A ) Schematic of key U2 snRNA secondary structure rearrangements in spliceosome assembly. Top: The 5′ sequence and nucleotide modifications of U2 snRNA as observed in the 17S U2 snRNP. The BSL stem and loop, toehold +1 nucleotides proposed for strand invasion and C28U cancer mutation are highlighted. The mutually exclusive Stem I extended is also shown. Bottom: In the catalytic spliceosome after branch helix formation, the 5′ end of U2 is completely remodeled to allow for base pairing with the intron and U6 snRNA. ( B ) Schematic of SV40 T antigen pre-mRNA highlights the different branchpoint sequences that can be used in the alternative splicing of large T and small t isoforms. ( C ) Comparison of the U2 snRNP interactions with large T and small t branchpoints with different combinations of exogenous U2 snRNA and splicing reporters. Details of base pairing between the U2 BPRS and small t branchpoint sequence with underlined branchpoint adenosine are included. The expected splice products for each combination are shown in the last column. Orthogonal nucleotides in U2 and the splicing reporter are highlighted. U2-WT* indicates the constant presence of endogenous U2 snRNA. ( D ) Primer extension analysis to quantify orthogonal U2 snRNA expression for the four transfection combinations shown in panel (C). Left: Representative PAGE of radiolabeled U2 snRNA primer extension reactions. Extension stops for endogenous U2 snRNA/U2-WT and U2-Ortho are indicated, along with positions of shark’s-tooth lane divisions. Right: Schematic of the primer extension reaction with the arrow representing the annealed radiolabeled primer and X’s marking the position of extension stops due to incorporation of ddGTP. ( E ) The fraction of U2-Ortho relative to total U2 snRNA quantified from primer extension reactions; n = 3, and ( F ) Levels of small t intron splicing as determined by -ΔΔCt analysis of RT-qPCR for the transfection combinations shown in panel (C). Briefly, -ΔΔCt was determined by first subtracting the Cq value for a small t splice junction probe from the Cq value of an exon 2 probe for each sample, and then from the ΔCt value for sample 2, making the -ΔΔCt value equivalent to log 2 -fold change in splicing efficiency relative to the transfection with BP-Ortho and U2-WT. In all cases, error bars represent standard deviation. ns = not significant, P -value <.05*, <.01**, <.005*** for Student’s t-test.
Article Snippet: Based on the system developed by Wu and Manley [ ], the SV40 Large T antigen gene was amplified from SV40 small + large T in pENTR1A (w611-7), a gift from Eric Campeau (Addgene plasmid #2229; http://n2t.net/addgene:22297; RRID:Addgene_22297 ), and subcloned into the AflII and NotI sites of pCI-M2 (a gift from John Olsen; Addgene plasmid #44170; http://n2t.net/addgene:44170; RRID:Addgene_44170 ) downstream of a CMV promoter.
Techniques: Sequencing, Mutagenesis, Alternative Splicing, Comparison, Expressing, Transfection, Quantitative RT-PCR, Standard Deviation
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