Journal: RNA

Article Title: Allele-specific SHAPE-MaP assessment of the effects of somatic variation and protein binding on mRNA structure

doi: 10.1261/rna.064469.117

Figure Lengend Snippet: The riboSNitches rs538915021 and rs11552475 alter the proportions of the 3′ UTR TPT1 mRNA structure clusters. ( A ) The SHAPE-guided structural ensemble for the reference sequence (control) is shown next to two riboSNitches (U850G and U867A). The riboSNitch U850G (rs538915021) causes an increase in population of the III cluster. The riboSNitch U867A (rs11552475) causes a decrease in the population of the I cluster. Three nonchangers are shown below (rs11540938, rs571864782, and rs751359423) that all occur within the same region. ( I–III ) Three representative medoid secondary structures are shown. Each cluster is labeled in the ensemble plots. The nucleotides in all structures are colored by WT SHAPE reactivity. ( B , C ) Comparison of normalized SHAPE data for the major and minor allele of the riboSNitches U850G (rs538915021) and U867A (rs11552475).

Article Snippet: For mutant analysis of the 3′ UTR of TPT1, HEK-293 cells were transfected with WT or mutant GFP-TPT1 3′ UTR constructs (GenScript, Clontech pTRE-TIGHT, NEB Q5 Site-Directed Mutagenesis Kit).

Techniques: Sequencing, Control, Labeling, Comparison

Journal: RNA

Article Title: Allele-specific SHAPE-MaP assessment of the effects of somatic variation and protein binding on mRNA structure

doi: 10.1261/rna.064469.117

Figure Lengend Snippet: TPT1 mRNA secondary structures are similar in the presence and absence of protein. ( A ) Median reactivity data from RNA in cellular lysate over a moving window of 40 nt indicating structured (low median SHAPE) and unstructured regions (high median SHAPE) within corresponds closely with the pattern of ( B ) median reactivity data from naked RNA. A 5′ to 3′ decrease in median SHAPE is observed, consistent with a highly structured 5′ UTR and less structured 3′ UTR. ( C ) Supportive of the overall similar median reactivities, pattern correlation of SHAPE reactivity is high (>0.85 overall). We performed correlations over multiple windows, from 10 to 50, bottom to top , with lighter blue indicating higher correlation. Regions of difference are noticeable as darker bands in the CDS and 3′ UTR. ( D ) TPT1 secondary structure model informed with SHAPE reactivies from unbound RNA modification. Regions of interest are labeled, including the start codon, stop codon, proposed PKR activating helices in the 5′ UTR, and putative AREs in the 3′ UTR.

Article Snippet: For mutant analysis of the 3′ UTR of TPT1, HEK-293 cells were transfected with WT or mutant GFP-TPT1 3′ UTR constructs (GenScript, Clontech pTRE-TIGHT, NEB Q5 Site-Directed Mutagenesis Kit).

Techniques: RNA modification, Labeling