Journal: Genes & Development
Article Title: AU-rich elements target small nuclear RNAs as well as mRNAs for rapid degradation
Figure Lengend Snippet: ARE-mediated HSUR 1 degradation in vivo. ( A ). ( B ) T1 RNase protection analysis of wild-type and mutant HSUR 1 levels in transient transfection assays. The pUC–U1–HSUR 1 constructs were transiently cotransfected with a pUC–U1–HSUR 3 plasmid into mouse L929 cells (see Materials and Methods). Total RNA collected 48 hr after transfection was subjected to RNase T1 protection assays with wild-type and mutant HSUR 1 antisense RNA (lanes 2 and 3, respectively), together with antisense HSUR 3 RNA as an internal control. One-fiftieth of the amount of the anti-wild-type and anti-mutant HSUR 1 RNA probes used is shown in lanes 4 and 5. The data were quantitated with a Molecular Dynamics PhosphorImager and normalized to HSUR 3. Wild-type HSUR 1 levels were reproducibly one-eighth of those of mutant HSUR 1. ( C ) Whole-cell run-on assays of wild-type and mutant HSUR 1 transcription. The pUC–U1–HSUR 1 construct containing wild-type or mutant HSUR 1 sequences was cotransfected with pUC–U1–HSUR 3 into L cells, and whole-cell run-on assays were performed (see Materials and Methods). Total RNA was hybridized to nylon membranes that had been dot-blotted with wild-type ( top ) or mutant ( bottom ) HSUR 1 and HSUR 3 DNA fragments. Untransfected HSUR 4 DNA was also dotted as a negative control. The patterns of dots are illustrated at right; hybridizations with the run-on RNAs are at left. After quantitation and normalization against the cotransfected positive control HSUR 3 (also subtracting the untransfected negative control HSUR 4), the wild-type HSUR 1 ( left dot, top ) and the mutant ( left dot, bottom ) were found to have similar transcription rates (wild type:mutant = 0.95). ( D ) Immunoprecipitation of wild-type and mutant HSUR 1 from transfected mouse L929 cells. L cells were transiently transfected with the pUC–U1 constructs containing wild-type or mutant HSUR 1 genes, and whole-cell extracts were prepared by sonication. Equal amounts of extract were precipitated with anti-Sm monoclonal antibody Y12 or anti-U1 70K monoclonal antibody H111 as a control. RNA was harvested from the immunoprecipitation pellets (lanes 1,3,5,7 ) and supernatants (lanes 2,4,6,8 ), and wild-type ( left ) and mutant ( right ) HSUR 1 were assayed by T1 RNase protection. For both wild-type and mutant HSUR 1s, > 90% of the RNA was in the anti-Sm precipitate (lanes 3,7 ), whereas > 99% of the HSUR 1s remained in the supernatant with the anti-U1 70K antibody (lanes 2,6 ).
Article Snippet: T1 RNase protection assays were performed as described (S. ), with the following modifications: DNase-treated RNA (5–10 μg for snRNA, 20–30 μg for mRNA) was combined with 2 × 105 to 4 × 105 cpm of the appropriate [α-32 P]UTP-labeled antisense probe, heated at 85°C for 5 min, incubated at 45°C overnight to allow annealing, and then digested with T1 RNase (1 U/10 μg of RNA; Calbiochem) at 30°C for 1 hr.
Techniques: In Vivo, Mutagenesis, Transfection, Construct, Plasmid Preparation, Negative Control, Quantitation Assay, Positive Control, Immunoprecipitation, Sonication