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. 2020 Nov 5;23(12):101780. doi: 10.1016/j.isci.2020.101780

Figure 5.

Figure 5

TBsRNA-33 Is an Anti-Sense Regulator of T. bruceiPif1 mRNA

(A) TBsRNA-33 interacts with Pif1 mRNA. (i) Schematic representation of the potential base-pairing between TBsRNA-33 and Pif1 mRNA. The arrows indicated the positions of primers used in (iii) to amplify the chimeric RNA. (ii) The sequence of chimeric RNA formed by the interaction between TBsRNA-33 and Pif1 mRNA 3′ UTR region is indicated from the PRS interactome library. (iii) Identification of TBsRNA-33 ligated to Pif1 mRNA 3′ UTR by RT-PCR. Total RNA was prepared using the protocol described in (Figure 3A), and the cDNA was subjected to gene specific PCR using a forward primer from TBsRNA-33, and reverse primers corresponding to different regions of Pif1 mRNA as indicated by the arrows in (i).

(B) TBsRNA-33 silencing. Cells carrying the silencing construct for TBsRNA-33 were silenced for the indicated days, and the RNA was subjected to Northern analysis with the indicated anti-sense probes.

(C) Pif1 mRNA is not increased following TBsRNA-33 silencing. Total RNA (20 μg) was prepared from cells before and after TBsRNA-33 silencing, separated on a 1.2% agarose/formaldehyde gel and subjected to Northern analysis using RNA probes, as indicated. 7SL RNA served as a loading control.

(D) PIF1 translation is affected upon TBsRNA-33 silencing. PIF1 was tagged N-terminus with MYC tag in cells carrying the silencing construct for TBsRNA-33 and were silenced for the indicated days. Whole cell lysate from induced (+TET) and uninduced (-TET) was subjected to Western analysis with the indicated antibodies (left). The expression level for PIF1-MYC is presented as mean ± S.E.M (right). Experiments were done in triplicate (n = 3). The expression level of HSP83 was used as a loading control. All densitometry data used in this figure were calculated by ImageJ (https://imagej.nih.gov/ij/) software.

(E) Dicistronic reporter cassette containing PIF1 3′ or 5′ UTR. tagRFPt gene and PIF1 UTR was cloned in PPOTv4 vector carrying eYFP gene (Dean et al., 2015).

(F) TBsRNA-33 regulate PIF1 expression via its 3′ UTR. Dicistronic reporter cassette was introduced in cells carrying the silencing construct for TBsRNA-33 and were silenced for the indicated days. Whole cell lysate from induced (+TET) and uninduced (-TET) cells were subjected to Western analysis with the indicated antibodies (left). The expression level for tagRFPt is presented as mean ± S.E.M (right). Experiments were done in duplicate (n = 2). The expression level of eYFP was used as a loading control. All densitometry data used in this figure was calculated by ImageJ (https://imagej.nih.gov/ij/) software.