A length-dependent evolutionarily conserved pathway controls nuclear export of circular RNAs

Abstract

Circular RNAs (circRNAs) are generated from many protein-coding genes. Most accumulate in the cytoplasm, but how circRNA localization or nuclear export is controlled remains unclear. Using RNAi screening, we found that depletion of the Drosophila DExH/D-box helicase Hel25E results in nuclear accumulation of long (>800-nucleotide), but not short, circRNAs. The human homologs of Hel25E similarly regulate circRNA localization, as depletion of UAP56 (DDX39B) or URH49 (DDX39A) causes long and short circRNAs, respectively, to become enriched in the nucleus. These data suggest that the lengths of mature circRNAs are measured to dictate the mode of nuclear export.

Keywords: DDX39A; DDX39B; Hel25E; UAP56; URH49; circRNA.

Figure 1.

Drosophila circRNAs are enriched in the cytoplasm but can accumulate in the nucleus upon depletion of Hel25E. (A) qRT–PCR quantification of endogenous Drosophila circRNAs in RNA purified from DL1 nuclear (Nuc) or cytoplasmic (Cyt) fractions. U6 and U7 snRNAs served as markers for the nuclear fraction. 18S ribosomal RNA (rRNA) and β-actin mRNA served as markers for the cytoplasmic fraction. The enriched fraction is set to 1. Data are shown as mean ± SD. n = 3. (B) DL1 cell lines stably expressing an inducible laccase2 (left) or dati (right) reporter plasmid were fractionated after 14 h of copper sulfate treatment to isolate nuclear and cytoplasmic RNA. Northern blots using 3 µg of each RNA fraction were then used to examine the localization of reporter-derived transcripts. (C,D) DL1 cells were treated with the indicated dsRNAs for 3 d followed by purification of nuclear (C) and cytoplasmic (D) RNA fractions. qRT–PCR was used to quantify expression of endogenous circlaccase2 and circdati. Data were normalized to the β-gal dsRNA sample and are shown as mean ± SD. n = 3. Known nuclear export receptors as well as components of the THO, TREX, and EJC are noted.

Figure 2.

Long (>800-nt) Drosophila circRNAs accumulate in the nucleus upon Hel25E depletion. (A,B) DL1 cells were treated for 3 d with a control (β-gal) dsRNA or a dsRNA to deplete Hel25E and then labeled with 250 µM 4sU for 15 min prior to isolating RNA from the nuclear (A) and cytoplasmic (B) fractions. 4sU-labeled nascent RNAs were purified from each fraction followed by qRT–PCR to quantify expression of the indicated circRNAs, which are ordered according to their mature circRNA lengths. Data were normalized to the β-gal dsRNA sample and are shown as mean ± SD. n = 3. (C) Reporter plasmids that produce firefly luciferase circRNAs (circfirefly) of different lengths were transfected into DL1 cells treated with the indicated dsRNAs. RNA was then purified from nuclear (N) or cytoplasmic (C) fractions, and 3.6 µg of each fraction was run on Northern blots to examine localization of reporter-derived transcripts.

Figure 3.

Localization of long and short human circRNAs is controlled by UAP56 and URH49, respectively. (A) qRT–PCR quantification of endogenous human circRNAs in RNA purified from HeLa nuclear (Nuc) or cytoplasmic (Cyt) fractions. MALAT1 and 18S rRNA served as markers for the nuclear and cytoplasmic fractions, respectively. The enriched fraction is set to 1. (B) Humans encode two closely related homologs of Drosophila Hel25E. The percent amino acid identity is shown. (C) HeLa cells were treated with siRNAs for 48 h to specifically deplete UAP56 or URH49. (D,E) HeLa cells were then fractionated to isolate nuclear (D) and cytoplasmic (E) RNA. qRT–PCR was used to quantify steady-state expression of the indicated circRNAs, which are ordered according to their mature RNA lengths. Data throughout the figure were normalized to the β-gal dsRNA samples and are shown as mean ± SD. n = 3. (**) P < 0.01; (*) P < 0.05.

Figure 4.

A region divergent among the Hel25E homologs controls their circRNA length preferences. (A) Domain structure of Hel25E family members, highlighting a four-amino-acid region that is divergent between human UAP56 and URH49. Expression plasmids were generated with these amino acids mutated to the indicated sequences. (B,C) DL1 cells were first treated with a control (β-gal) dsRNA or a dsRNA to deplete endogenous Hel25E. On the next day, inducible plasmids expressing wild-type (WT) or mutant (MUT) helicases (insensitive to dsRNA treatment) were transfected. Copper sulfate (500 μM) was added after 8 h to induce helicase expression from the plasmids, and cells were fractionated after 24 h. qRT–PCR was then used to quantify steady-state expression of endogenous circdati (B) or circEct4 (C) in nuclear and cytoplasmic fractions. Data were normalized to the β-gal dsRNA sample and are shown as mean ± SD. n = 3. (**) P < 0.01; (*) P < 0.05.