Journal: RNA Biology

Article Title: A Gypsy element contributes to the nuclear retention and transcriptional regulation of the resident lncRNA in locusts

doi: 10.1080/15476286.2021.2024032

Figure Lengend Snippet: The Gypsy element prolongs the life-span of PAHAL . (a) The embedded Gypsy element contributes to the predicted lncRNA secondary structure. ‘MFE’ is the minimum free energy. The yellow line indicates the region of the embedded Gypsy element. (b) RNA stability of PAHAL and the Gypsy element-deleted PAHAL (i.e. PAHAL Δ Gypsy ). The SRSF2 protein-depleted mouse embryonic fibroblast line (SRSF2-MEFs) that overexpressed PAHAL or PAHAL Δ Gypsy was treated with the transcriptional inhibitor actinomycin D (Act D) or vehicle (0.1% DMSO) for 1 to 7 h. PAHAL and PAHAL Δ Gypsy levels were measured using RT-qPCR (N = 7). (c) Decay of PAHAL and PAHAL¯ RNA in the presence of Act D at the brain. The insets show parameters for the fitted curves using one-phase exponential decay. Eight replicates of eight brains were measured. Student’s t test: * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant.

Article Snippet: Different constructs were prepared: The full-length PAHAL¯ sequence (labelled as PAHAL¯ ), PAHAL with the Gypsy retroelement deleted (i.e. PAHAL Δ Gypsy ), PAHAL with the NLS deleted ( PAHAL ΔNLS ), the PAHAL¯ sequence with an artificial insertion of the Gypsy element immediately preceding the poly(A) sequence of PAHAL¯ (i.e. PAHAL¯ Gypsy+ ), and PAHAL with the mutation of the tandem ESEs (i.e. MT-ESEs- PAHAL ); they were cloned into the pcDNA3.1 (+) vector (overexpression vector for SRSF2-MEFs; Invitrogen, CA, USA; V79020) and pAc5.10/V5-His A vector (overexpression vector for Drosophila S2 cells; Invitrogen, CA, USA; V4110-20). pGL4.10-P + 5′UTR (with the PAH promoter fused to a firefly luciferase reporter), pcDNA3.1/ PAHAL ( PAHAL overexpression vector for SRSF2-MEFs), pAc5.10/V5-His A/ PAHAL ( PAHAL overexpression vector for Drosophila S2 cells), pcDNA3.1/ lacz (negative control vector for SRSF2-MEFs), pAc5.10/V5-His A/ lacz (negative control vector for Drosophila S2 cells), pcDNA3.1/V5-His/ SRSF2 ORF (SRSF2 overexpression vector for SRSF2-MEFs) and pAc5.10/V5-His A/ SRSF2 ORF (SRSF2 overexpression vector for Drosophila S2 cells) were constructed and described previously [ ].

Techniques: Quantitative RT-PCR

Journal: RNA Biology

Article Title: A Gypsy element contributes to the nuclear retention and transcriptional regulation of the resident lncRNA in locusts

doi: 10.1080/15476286.2021.2024032

Figure Lengend Snippet: The Gypsy element promotes the nuclear retention of PAHAL . (a) The red bold text represents the nuclear location signal (NLS) sequence. The conserved nucleotides are highlighted using an underscored line. The boldface with blue shading indicates the three predicted SRSF2 high-affinity sites (ESEs) in the Gypsy element embedded in PAHAL , which are labelled ESE1, ESE2 and ESE3. ‘+2441’, ‘+2496’ and ‘+2602’ represent the start sites of the three ESEs relative to the transcript start site of PAHAL . The yellow characters represent the mutant sequence of the ESEs. (b) Nuclear localization of PAHAL and PAHAL¯ in the locust brains. U6 and β-actin , stable and abundant housekeeping genes that localize to the nucleus and cytoplasm in the locust brains, respectively, were used as internal controls to test the localization of PAHAL and PAHAL¯ in the brain. Five biological replicates of twenty brains were examined for each nuclear fractionation. (c) Nucleocytoplasmic shuttling of four PAHAL RNA variants, i.e. wild-type PAHAL, PAHAL¯ with insertion of the Gypsy element into its 3′ end (labelled as PAHAL¯ Gypsy+ ), PAHAL with Gypsy deletion (i.e. PAHAL Δ Gypsy ), and NLS-deleted PAHAL (i.e. PAHAL ΔNLS ) in SRSF2-MEFs (N = 6). U2 and β-actin , indicate the nucleus and cytoplasm in SRSF2-MEF, respectively, were used to determine if there were specific changes in the pattern of PAHAL localization elicited by Gypsy or NLS deletion. (d) FISH detection of the subcellular localization of PAHAL, PAHAL Δ Gypsy and PAHAL ΔNLS . Images are shown at 63× magnification, and scale bars represent 10 µM. The arrows indicate the subcellular location of PAHAL (in the nucleus), PAHAL Δ Gypsy and PAHAL ΔNLS (in the cytoplasm). (e) The half-life of PAHAL¯ Gypsy+ and PAHAL ΔNLS RNA in the presence of Act D at SRSF2-MEF. Six replicates were measured. Student’s t test: * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant.

Article Snippet: Different constructs were prepared: The full-length PAHAL¯ sequence (labelled as PAHAL¯ ), PAHAL with the Gypsy retroelement deleted (i.e. PAHAL Δ Gypsy ), PAHAL with the NLS deleted ( PAHAL ΔNLS ), the PAHAL¯ sequence with an artificial insertion of the Gypsy element immediately preceding the poly(A) sequence of PAHAL¯ (i.e. PAHAL¯ Gypsy+ ), and PAHAL with the mutation of the tandem ESEs (i.e. MT-ESEs- PAHAL ); they were cloned into the pcDNA3.1 (+) vector (overexpression vector for SRSF2-MEFs; Invitrogen, CA, USA; V79020) and pAc5.10/V5-His A vector (overexpression vector for Drosophila S2 cells; Invitrogen, CA, USA; V4110-20). pGL4.10-P + 5′UTR (with the PAH promoter fused to a firefly luciferase reporter), pcDNA3.1/ PAHAL ( PAHAL overexpression vector for SRSF2-MEFs), pAc5.10/V5-His A/ PAHAL ( PAHAL overexpression vector for Drosophila S2 cells), pcDNA3.1/ lacz (negative control vector for SRSF2-MEFs), pAc5.10/V5-His A/ lacz (negative control vector for Drosophila S2 cells), pcDNA3.1/V5-His/ SRSF2 ORF (SRSF2 overexpression vector for SRSF2-MEFs) and pAc5.10/V5-His A/ SRSF2 ORF (SRSF2 overexpression vector for Drosophila S2 cells) were constructed and described previously [ ].

Techniques: Sequencing, Mutagenesis, Fractionation

Journal: RNA Biology

Article Title: A Gypsy element contributes to the nuclear retention and transcriptional regulation of the resident lncRNA in locusts

doi: 10.1080/15476286.2021.2024032

Figure Lengend Snippet: The embedded Gypsy element regulates the tethering of PAHAL with SRSF2. (a) RNA immunoprecipitation (RIP) in SRSF2-MEFs verified the effect of the embedded Gypsy element on the binding of SRSF2 with PAHAL . (b) RIP was performed in locust brains to test the binding affinity of SRSF2 to PAHAL¯ RNA in vivo . Six biological replicates of fifty brains were examined. (c) Deletion of the NLS did not affect the SRSF2 binding affinity of PAHAL . (d) Mutational analysis of the SRSF2 affinity of the ESEs in the Gypsy element of PAHAL . Wild-type and mutant probes were incubated with SRSF2-MEF lysates that overexpressed the V5-tagged SRSF2 (SRSF2-V5) of locusts. MT-ESEs indicates the mutation of all three ESEs. MT-ESE1, MT-ESE2 or MT-ESE3 indicates mutated ESE1, ESE2 or ESE3, respectively. WT indicates wild type. (e) The predicted lncRNA secondary structure altered by mutation of ESEs in the Gypsy element. MT-ESEs- PAHAL means PAHAL with the mutation of ESEs in the Gypsy element. The blue shading indicates the ESEs of the embedded Gypsy element (indicated by the yellow line) in PAHAL . (f) The life time of MT-ESEs- PAHAL in the SRSF2-MEF adding Act D. (g) SRSF2 is not required for the nuclear retention of PAHAL . The following vectors were transfected into SRSF2-MEFs that had been treated using DOX for 1 day to knock out endogenous SRSF2: ‘SRSF2+’, pcDNA3.1/V5-His/ SRSF2 ORF; ‘SRSF2−’, pcDNA3.1/V5-His/ lacz ; ‘ PAHAL +’, pcDNA3.1(+)/ PAHAL ; ‘ PAHAL −’, pcDNA 3.1(+)/ lacz ; ‘ PAHAL Δ Gypsy +’, pcDNA3.1(+)/ PAHAL Δ Gypsy ; ‘ PAHAL Δ Gypsy −’, pcDNA 3.1(+)/ lacz . Five replicates were measured. The different letters within each column indicate that the means are significantly different ( P < 0.05).

Article Snippet: Different constructs were prepared: The full-length PAHAL¯ sequence (labelled as PAHAL¯ ), PAHAL with the Gypsy retroelement deleted (i.e. PAHAL Δ Gypsy ), PAHAL with the NLS deleted ( PAHAL ΔNLS ), the PAHAL¯ sequence with an artificial insertion of the Gypsy element immediately preceding the poly(A) sequence of PAHAL¯ (i.e. PAHAL¯ Gypsy+ ), and PAHAL with the mutation of the tandem ESEs (i.e. MT-ESEs- PAHAL ); they were cloned into the pcDNA3.1 (+) vector (overexpression vector for SRSF2-MEFs; Invitrogen, CA, USA; V79020) and pAc5.10/V5-His A vector (overexpression vector for Drosophila S2 cells; Invitrogen, CA, USA; V4110-20). pGL4.10-P + 5′UTR (with the PAH promoter fused to a firefly luciferase reporter), pcDNA3.1/ PAHAL ( PAHAL overexpression vector for SRSF2-MEFs), pAc5.10/V5-His A/ PAHAL ( PAHAL overexpression vector for Drosophila S2 cells), pcDNA3.1/ lacz (negative control vector for SRSF2-MEFs), pAc5.10/V5-His A/ lacz (negative control vector for Drosophila S2 cells), pcDNA3.1/V5-His/ SRSF2 ORF (SRSF2 overexpression vector for SRSF2-MEFs) and pAc5.10/V5-His A/ SRSF2 ORF (SRSF2 overexpression vector for Drosophila S2 cells) were constructed and described previously [ ].

Techniques: Immunoprecipitation, Binding Assay, In Vivo, Mutagenesis, Incubation, Transfection, Knock-Out

Journal: RNA Biology

Article Title: A Gypsy element contributes to the nuclear retention and transcriptional regulation of the resident lncRNA in locusts

doi: 10.1080/15476286.2021.2024032

Figure Lengend Snippet: The Gypsy element embedded in PAHAL is required for PAHAL- mediated transcription activation. (a) PAHAL¯ inhibits the promoter activity of PAH . ‘P + 5′UTR’ contains −1,168 to +89 bp relative to the TSS, representing the PAH promoter (‘P’: −1,168 to +1 bp) and 5′- untranslated region (‘5′UTR’: +1 to +89 bp). (b) The Gypsy element is required for the regulatory function of PAHAL . ‘ Lacz ’ is a frameshift mutational fragment of the lacz gene and serves as a negative control. PAHAL, PAHAL¯, PAHAL Δ Gypsy and lacz were inserted into the pAC5.1/V5-His A vector, which is a high-level transient expression plasmid in Drosophila S2 cells. (c) The embedded Gypsy element participates in the interaction of SRSF2 and PAHAL for PAH transcription activation. SRSF2-MEFs (SRSF2-KO) were used to test the activity of the P + 5′-UTR by luciferase assays. ‘DOX +’ represents endogenic SRSF2 knockout, whereas ‘DOX – ’ or ‘Control’ indicates the normal expression of endogenic SRSF2 in SRSF2-MEFs or in S2 cells. ‘SRSF2-V5’ indicates the overexpression of the V5-tagged locust SRSF2 in S2 cells (S2, SRSF2-ov). Student’s t test: *P < 0.05; **P < 0.01; ***P < 0.001; n.s., nonsignificant. (d) The ESEs and NLS in the Gypsy of PAHAL contributes to the PAHAL -mediated transcription activation of PAH . Six replicates were measured. The different letters within each treatment indicate that the means are significantly different ( P < 0.05).

Article Snippet: Different constructs were prepared: The full-length PAHAL¯ sequence (labelled as PAHAL¯ ), PAHAL with the Gypsy retroelement deleted (i.e. PAHAL Δ Gypsy ), PAHAL with the NLS deleted ( PAHAL ΔNLS ), the PAHAL¯ sequence with an artificial insertion of the Gypsy element immediately preceding the poly(A) sequence of PAHAL¯ (i.e. PAHAL¯ Gypsy+ ), and PAHAL with the mutation of the tandem ESEs (i.e. MT-ESEs- PAHAL ); they were cloned into the pcDNA3.1 (+) vector (overexpression vector for SRSF2-MEFs; Invitrogen, CA, USA; V79020) and pAc5.10/V5-His A vector (overexpression vector for Drosophila S2 cells; Invitrogen, CA, USA; V4110-20). pGL4.10-P + 5′UTR (with the PAH promoter fused to a firefly luciferase reporter), pcDNA3.1/ PAHAL ( PAHAL overexpression vector for SRSF2-MEFs), pAc5.10/V5-His A/ PAHAL ( PAHAL overexpression vector for Drosophila S2 cells), pcDNA3.1/ lacz (negative control vector for SRSF2-MEFs), pAc5.10/V5-His A/ lacz (negative control vector for Drosophila S2 cells), pcDNA3.1/V5-His/ SRSF2 ORF (SRSF2 overexpression vector for SRSF2-MEFs) and pAc5.10/V5-His A/ SRSF2 ORF (SRSF2 overexpression vector for Drosophila S2 cells) were constructed and described previously [ ].

Techniques: Activation Assay, Activity Assay, Negative Control, Plasmid Preparation, Expressing, Luciferase, Knock-Out, Over Expression

Journal: RNA Biology

Article Title: A Gypsy element contributes to the nuclear retention and transcriptional regulation of the resident lncRNA in locusts

doi: 10.1080/15476286.2021.2024032

Figure Lengend Snippet: Working model for the embedded Gypsy element-mediated lncRNA transcriptional regulation. The Gypsy element embedded in PAHAL is required for the nuclear retention and RNA stability of PAHAL . Instead, the lncRNA without the element, i.e. PAHAL¯ , is exported to the cytoplasm for decay. The embedded Gypsy element boosts the recruitment of SRSF2 to form an RNA-protein complex, which is crucial for the PAHAL -mediated promoter activation of PAH . The red genomic region in the PAH locus represents the insertion site of the Gypsy element.

Article Snippet: Different constructs were prepared: The full-length PAHAL¯ sequence (labelled as PAHAL¯ ), PAHAL with the Gypsy retroelement deleted (i.e. PAHAL Δ Gypsy ), PAHAL with the NLS deleted ( PAHAL ΔNLS ), the PAHAL¯ sequence with an artificial insertion of the Gypsy element immediately preceding the poly(A) sequence of PAHAL¯ (i.e. PAHAL¯ Gypsy+ ), and PAHAL with the mutation of the tandem ESEs (i.e. MT-ESEs- PAHAL ); they were cloned into the pcDNA3.1 (+) vector (overexpression vector for SRSF2-MEFs; Invitrogen, CA, USA; V79020) and pAc5.10/V5-His A vector (overexpression vector for Drosophila S2 cells; Invitrogen, CA, USA; V4110-20). pGL4.10-P + 5′UTR (with the PAH promoter fused to a firefly luciferase reporter), pcDNA3.1/ PAHAL ( PAHAL overexpression vector for SRSF2-MEFs), pAc5.10/V5-His A/ PAHAL ( PAHAL overexpression vector for Drosophila S2 cells), pcDNA3.1/ lacz (negative control vector for SRSF2-MEFs), pAc5.10/V5-His A/ lacz (negative control vector for Drosophila S2 cells), pcDNA3.1/V5-His/ SRSF2 ORF (SRSF2 overexpression vector for SRSF2-MEFs) and pAc5.10/V5-His A/ SRSF2 ORF (SRSF2 overexpression vector for Drosophila S2 cells) were constructed and described previously [ ].

Techniques: Activation Assay