Fig. 1: Functional and structural investigation of the interplay of UPF1 with the histone SL. | Nature Communications

Fig. 1: Functional and structural investigation of the interplay of UPF1 with the histone SL.

From: Mechanistic insights into recruitment and regulation of the RNA helicase UPF1 in replication-dependent histone mRNA decay

Fig. 1: Functional and structural investigation of the interplay of UPF1 with the histone SL.The alternative text for this image may have been generated using AI.

A Domain organization of human UPF1 and 3′hExo. UPF1 variants used in this study are shown below the schematic. The sequence of the histone SL and variants are indicated. B Unwinding activity of UPF1-Hel on substrates containing or lacking the histone SL (SL and linear RNA, respectively). Top: experimental setup of the nucleic acid-unwinding assay. Bottom: Time-dependent measurements of UPF1-Hel unwinding activity on the linear (white) and SL RNA (black) substrates. Data represent the mean of 3 independent experiments, with technical duplicates for every experiment. Shaded areas represent the standard deviation. An unpaired two-tailed t-test (p = 0.107) indicates that the difference between unwinding of the linear and SL-RNA substrate is not significant (denoted by ns). The data were fitted to a two-state decay model to obtain the first-order rate constants, kfast and kslow. C Time-dependent analysis of degradation of 60N-SL19 RNA by 3′hExo in the absence and presence of UPF1-Hel. Addition of UPF1 increases the efficiency (rate and progression) of the degradation reaction, generating a pattern that is comparable to addition of 2x-3′hExo. Densitometric analysis on the urea-PAGE gel is shown in the right panel. The columns and error bars of the plot represent mean values and standard deviation derived from analysis of 3 independent experiments (shown as data points). D CryoEM reconstruction of UPF1-CHh:U-SL RNA. Left: density map (gray) obtained from cryoEM analysis with individual domains of UPF1 (colored according to the schematic in A) modeled in. Middle: 3D reconstruction colored by domains as in (A). The additional density is colored black. Right: Schematic of the U-SL RNA. The SL was modeled into the density indicated by a black arrow. E Structural overview of UPF1-CHh bound to the U-SL RNA. UPF1-CHh is shown in a surface representation and the U-SL RNA as a cartoon. The 5′ single stranded stretch of the RNA threads into the UPF1-RNA binding channel while the SL is positioned at the entry site of the channel formed by domains 1B, 1C and the stalk helices (shown in gray).

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