Small RNA expression and deep sequencing analyses of the nucleolus reveal the presence of nucleolus-associated microRNAs

2.1 Cell line and reagents

HeLa cervical adenocarcinoma cells (CCL-2, ATCC) and HCT116 cells (wt and DICER−/−) were maintained in DMEM and 10% FCS. The HCT116 DICER−/− cells were a kind gift of Dr. Velculescu (Johns Hopkins University, Baltimore, MD). MCF7 breast adenocarcinoma cells (HTB22, ATCC) were maintained in MEM supplemented with 10% FCS and non-essential amino acids. All cell culture reagents were obtained from Invitrogen. The following drugs were used; Actinomycin D (Sigma–Aldrich), Leptomycin B (Sigma–Aldrich).

2.2 Fractionation of cells and isolation of RNA

Nuclear, nucleolar and cytoplasmic fractions were prepared essentially as in our previous study [45]. RNA was extracted from subcellular compartments using TRIzol Reagent (Invitrogen). From a starting population of about 1.8 × 10⁸ cells, 6% of the nuclei were used for extraction of nuclear RNA (typically yielding 120 μg RNA), and the rest (94%) was used for extraction of nucleoli and nucleolar RNA (typically yielding 40–160 μg RNA). Of the cytoplasmic fraction corresponding to 1.8 × 10⁸ cells, 1.6% was used for extraction of cytoplasmic RNA (typically yielding 240 μg RNA). This leads to a RNA output ratio of 1:10:80 per nucleolus/nucleus/cytoplasm.

2.3 NCode™ microRNA profiling

Total RNA was extracted from whole cells and nucleoli using the TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. Small RNAs (<200 nt) were further enriched using mirVana kit (Ambion). The small RNAs were poly-A tailed and tagged with the sequence tag for either Alexa Fluor® 5 fluorophor or Alexa Fluor® 3 fluorophor using the NCode™ miRNA Labeling System (Invitrogen). The samples were hybridized in triplicate to NCode™ Multi-Species miRNA Microarray (Invitrogen) overnight according to the instructions by the manufacturer. The microarrays were then hybridized with Alexa Fluor® 3 and Alexa Fluor® 5 capture reagents and washed. Hybridization was performed on a MAUI hybridization station (BioMicro Systems Inc). Each array was subsequently scanned using an Agilent DNA microarray scanner, and images were processed using GenePix Pro software (Molecular Devices). Data analyses were conducted using R statistical program. Nucleolar and cellular miRNA intensities were normalized according to intensities of 6–7 snoRNAs included in the arrays. HeLa and MCF7 cellular intensities were normalized by fold difference of their averaged cellular intensity.

2.4 Heat-treatment of nucleoli and quantitative RT-PCR

Equal amount of freshly purified HeLa nucleolus suspension was either heated to 95 °C (“heat-denatured nucleolus”) for 5 min or incubated on ice for 5 min (intact nucleolus). The nucleoli were then subjected directly to reverse transcription using SuperScript® III Reverse Transcriptase (Invitrogen) and random hexamers according to the manufacturer's instructions. QPCR was performed using SYBR Green (Atila Biosystem) and specific primer pairs. Amplification was conducted for 38 cycles at 94 °C for 10 s and at 60 °C for 1 min each using an ABI7900 thermocycler (Applied Biosystems). ΔCt represents Ct heat-denaturated nucleoli – Ct non-denatured nucleoli. The primer sequences used were as follows: RNU44: 5′-CCTGGATGATGATAGCAAATGC-3′ and 5′-GAGCTAATTAAGACCTTCATGTT-3′; RNU48: 5′-AGTGATGATGACCCCAGGTAA-3′ and 5′-GTGATGGCATCAGCGACACA-3′; HBII-239: 5′-GAAGCAGTGGGAGTGGAGAA-3′ and 5′-TCAGCAGTTTGAGTGTCAGCA-3′; hY1: 5′-GGCTGGTCCGAAGGTAGTGA-3′ and 5′-GCAGTAGTGAGAAGGGGGGA-3′; hY3: 5′-GGCTGGTCCGAGTGCAGTG-3′ and 5′-GAAGCAGTGGGAGTGGAGAA-3′; U1: 5′-ATACTTACCTGGCAGGGGAGA-3′ and 5′-CAGGGGGAAAGCGCGAACGCA-3′; U2: 5′-ATCGCTTCTCGGCCTTTTTG-3′ and 5′-TCCTATTCCATCTCCCTGCTC-3′. Primers for miR-206, miR-23a and miR-100 were from Applied Biosystems (TaqMan miRNA Assays ID 000510, ID 000399 and ID 000437).

2.5 TaqMan microRNA array

Comparisons of global miRNA expression profiles in intact and heat-treated nucleoli were performed using the TaqMan Array Human MicroRNA Panel v2.0 (Applied Biosystems), which includes Cards A and B in a 384-well format. These contain assays for quantification of 664 human miRNAs and 7 control RNAs. The experimental procedures were according to the manufacturer's instructions. In brief, suspension of intact and heat-treated nucleoli were directly subjected to reverse-transcription using the Multiplex RT pool set (Applied Biosystems) and the High-Capacity cDNA Archive Kit (Applied Biosystems), whereby a stem-loop RT primer specifically binds to its corresponding miRNA and initiates its reverse-transcription. The reaction products were subsequently amplified with sequence-specific primers using the Applied Biosystems 7900 HT Real-Time PCR system. The reactions were incubated in a 384-well plate at 95 °C for 10 min followed by 40 cycles at 95 °C for 15 s and at 60 °C for 1 min. The data were collected and processed using the Plate Utility and Automation Controller software (Applied Biosystems).

2.6 RNA isolation and generation of cDNA libraries

Total RNA was extracted from whole cells and the isolated cellular compartments using TRIzol Reagent according to manufacturer's instructions as outlined in our previous study [45] and manuscript submitted. Approximately 400 μg total RNA from each compartment was used to prepare cDNA libraries. Briefly, small RNAs (less than 200 nt) were isolated from the total RNA samples using mirVana Isolation Kit (Ambion) and separated on denaturing 15% polyacrylamide gel (15% PAA, 19:1 acrylamide/bis, 7 M urea) to purify RNAs in the approximate range of 10–40 nt. The small RNA size range was confirmed and the RNA was quantified using Bioanalyzer and the Agilent Small RNA kit (Agilent). cDNA libraries were constructed using the Ion Torrent RNA-seq kit v1 for small RNA libraries according to manufacturer's instructions, and their purity and concentration was confirmed using Bioanalyzer with the Agilent DNA kit. Small RNA library sequencing was performed using a 314 chip on an Ion Torrent sequencing platform (Life Technologies, Invitrogen) at the Sidney Kimmel Comprehensive Cancer Center Next Generation Sequencing Core. Raw data is deposited to GEO: GSE50057.

2.7 Northern hybridization

Northern blotting was carried out according to Ref. [12] with some modifications. Briefly, 20 μg RNA were resolved on denaturing polyacrylamide gels in 1X TBE, transferred onto a positively charged nylon membrane (Roche Diagnostics) using semi-dry electroblotting (Bio-Rad), and immobilized by UV irradiation (Stratagene Crosslinker 120 mJ/cm²). The membrane was pre-hybridized with hybridization buffer at 37 °C for 1 h, and then hybridized overnight at 37 °C with specific oligodeoxynucleotides. The probes were labeled with Digoxigenin using the DIG oligonucleotide 3′END labeling kit (Roche) and detected using the DIG Nucleic Acid Detection Kit (Roche). The following probes were used: RNU44: 5′-AGTTAGAGCTAATTAAGACCT-3′; tRNA-Ile, 5′-UGGUGGCCCGUACGGGGAUCGA-3′; U11: 5′-TCTTGATGTCGATTCCGCACGCAGAGCAATCGAGTTGCCC-3′; hY1: 5′-AAGGGGGGAAAGAGTAGAACA-3′. The probes were synthesized at the Johns Hopkins Genetic Resources Core Facility. The miRCURY locked nucleic acid (LNA) Digoxigenin-labeled probes for detection of miR-19b, miR-21, miR-23a, miR-24, miR-30c, miR-31, miR-99a and miR-191 were purchased from Exiqon.

3.1 MiRNA expression arrays of nucleolar RNA

We conducted miRNA expression profiling of HeLa cervical and MCF7 breast adenocarcinoma cells. For this purpose, we isolated nucleoli and purified small RNAs from the nucleolar preparation and from total cellular RNA. The nucleoli were visually verified for 99% purity under phase contrast and by staining the nucleoli for nucleolar marker protein nucleophosmin (not shown). Analysis of small RNAs by 16% denaturing PAGE showed that the RNAs were distinct (Fig. 1 A). The RNAs were dual-color labeled and hybridized to NCode Multispecies miRNA array v.2 using three technical repeats. Signals of over 60% miRNAs present in the array were detected in both cell lines. The nucleolar signal intensities were normalized according to several snoRNAs present in the array (e.g. U46, U47, U49, U50, HBII-13, HBII-239 and HBII-436), and data for high-ranking miRNAs are shown in Supplementary Table S1. As shown in Fig. 1B and C, high-intensity signals were detected in both cell lines in the total cellular and nucleolar RNA preparations. Scatter plot graphs showed that cellular miRNA content correlated with nucleolar signal intensity (Pearson correlation coefficiency, r = 0.843) in HeLa cells, whereas modest correlation was observed in MCF7 cells (r = 0.567). In HeLa and MCF7 nucleolar RNA isolates, 78% of top-ranking 46 miRNAs were identical, and their intensities were highly associated (Fig. 1D and E). However, the intensities of cellular miRNAs in the cell lines were not (Fig. 1F). Data derived from HeLa cells was suggestive that highly expressed miRNAs were detected also in the nucleolar RNA preparation.

However, cellular RNAs present in the nucleolar preparation may confound this interpretation. We hence decided to test an approach that benefits from the highly compact structure of the nucleolus. We reasoned that if non-nucleolar RNAs are present in the nucleolar preparation there would be a substantial difference in the detection rates if isolated, intact nucleoli as compared to nucleoli subjected to denaturing conditions are used as source for miRNA profiling. For this and subsequent analyses we decided to use HeLa cells due to the abundance of miRNAs present, and our previous comparative analyses on cellular and nucleolar proteins and RNA in this cell line [39, 45]. Accordingly, we subjected isolated HeLa nucleoli to heat-denaturation (95 °C, 5 min) or not, followed by qPCR using TaqMan assay designed for detection of mature miRNAs (Fig. 2 A). Analysis of abundant, largely cytoplasmic RNAs (hY1, hY3) or nuclear RNAs (U1, U2) showed that there was no difference in the amplification cycles (Ct) between the denatured vs. non-denatured nucleoli (ΔCt), whereas the ΔCt of snoRNAs RNU44 and RNU48 were close to 6 (Fig. 2B). In a further repeat of this assay, RNU44 and RNU48 had ΔCt-values of ⩾7, representing over 128-fold higher detection rate in the denatured nucleolar sample (Fig. 2C). Of three miRNAs tested in the same assay, ΔCt of miR-100 was similar to the snoRNAs (ΔCt 7.8), and miR-23a was somewhat lower (ΔCt 5.4) (Fig. 2C). There was no change in miR-206, included into the analysis based on a previous publication [16], but which was present in HeLa at very low level (Fig. 2C). We concluded that this approach provided a feasible way to enrich and detect nucleolus-associated small RNAs.

We then conducted TaqMan miRNA array profiling of isolated nucleoli using the comparative heat-denaturation protocol. To further increase robustness, we limited the amplification cycles to <38 to exclude rare and potentially contaminating signals. Of a total of 664 miRNAs represented by the array, 92 individual miRNAs were detected (Fig. 3 A). Of these, 32 miRNAs had ΔCt ⩾ 7, that is, were 128-fold more abundant in the heat-denatured nucleolus (Fig. 3B). All snoRNA controls included in the array had ΔCt of over 10, whereas small nuclear RNAs had <4 (Fig. 3B). Based on these results we believe that the assay represents with good predictability the presence of miRNAs in the nucleolar RNA preparation. The identified nucleolar miRNAs included members of four miRNA families, namely miR-17-92, miR-106a-363, miR-23a-24 and miR-30 (Fig. 3C). This may be an indicator that these miRNAs share regulatory pathways or functional activities relevant for their nucleolar presence.

3.2 Northern hybridization of miRNA in subcellular compartments

To ascertain the presence of miRNAs in the nucleolar RNA preparations we conducted Northern hybridization of seven representative miRNAs. We assessed the presence of the miRNAs in the nucleolar, nuclear, cytoplasmic and total cellular fractions. Cellular fractionation and RNA isolation was performed as in our previous studies [39, 45] and manuscript submitted (Bai et al., submitted). Equal amounts of RNA (25 μg) was loaded from each subcellular fraction. The purity of the RNA preparations was confirmed using probes against several small RNAs. These included RNU44, a snoRNA abundantly expressed in the nucleolus, U11, a small nuclear RNA with mostly nucleoplasmic localization, cyto-nucleoplasmic tRNA, and hY1, a mostly cytoplasmic RNA. According to the Northern analysis, and analyses shown in Refs. [39, 45], the nucleolar RNA preparations were judged to be free of nucleoplasmic and cytoplasmic RNA (Fig. 4 A). To further test the purity of RNA isolates we conducted qPCR using primers for cytoplasmic hY1 and nucleolar RNU44. The qPCR for hY1 and RNU44 confirmed their enrichment in the compartment-specific RNA preparations (Fig. 4C). As shown in Fig. 4B, several miRNAs (miR-19b, miR-21, miR-24, miR-30c, miR-31, miR-99a, miR-191) were detected in the nucleolar fraction in their mature 20–23 nt forms, but were proportionally much lower as compared to the total cellular RNA fraction. Mature miRNAs were also detected in the nuclear RNA fraction. This is consistent with recent deep sequencing of small RNAs, which showed nuclear presence of over 60 mature miRNAs including all miRNAs analyzed here [13].

3.3 Small RNA deep sequencing of the subcellular compartments

To provide a comprehensive and quantitative analysis of miRNA abundance in the HeLa subcellular fractions, we used small RNA deep sequencing. Following subcellular fractionation, RNA isolation and separation, small RNAs (10–40 nt) were gel purified and used to generate cDNA libraries followed by deep sequencing using Ion Torrent platform. Full data set is deposited as #GSE50057, and detailed description of the small RNA annotation is provided in a manuscript submitted (Bai et al., submitted). The analysis showed that the nucleolar small RNome was unique and predominantly composed of snoRNA-derived small RNAs. Approximately 200,000 miRNA reads were detected in the total cellular, cytoplasmic and nuclear fraction each, and 2100 reads in the nucleolar RNA library (Table 1 and Supplementary Table S2). Eleven miRNAs were present in the nucleolar small RNA fraction at read numbers ⩾10. Of these, eight were present in the TaqMan array (miR-19b, miR-21, miR-24, miR-31, miR-93, miR-151, miR-532, miR-574) and scored positive in the nucleolus-denaturation protocol (Fig. 3B). Overall, the miRNA reads between cytoplasmic and total RNA and nuclear and total RNA showed linear distribution as displayed in scatter plots, and very high correlation (Pearson correlation coefficiency 0.998 and 0.997, respectively) (Supplementary Fig. S1A and B). Scatter plot analyses of the distribution between nucleolar and cytoplasmic or nuclear miRNAs were more diverse, although a general trend of correlation between the read abundances was maintained (Supplementary Fig. S1C and D). The Pearson correlations between nucleolar and cytoplasmic or nuclear reads were 0.634 and 0.735, respectively.

Given that small RNA deep sequencing of cytoplasmic and nuclear fractions have indicated that mature miRNAs are frequently present in the nucleus consistent with our results here, we queried for this property by comparing reads of all miRNAs present in both compartments at reads ⩾10. For this purpose, we normalized the numbers of nuclear and cytoplasmic reads against the cellular reads, and then compared the relative ratios of nuclear and cytoplasmic reads. 17 miRNAs were 2-fold more abundant in the nucleus than in the cytoplasm (Fig. 5 A), and included e.g. miR-29b previously shown to be stabilized in HeLa cell nuclei [12]. Conversely, those miRNAs with higher relative cytoplasmic abundances included seven members of miR-let-7 family and miR-34a (Fig. 5A).

We then compared the relative frequencies of the eleven nucleolar miRNA reads to their frequencies in the other cellular fractions. This analysis showed that the frequencies of several miRNAs were substantially higher in the nucleolus than in the nuclear and cytoplasmic compartments (Fig. 5B). MiR-574, miR-1307, miR-31, miR-148, miR-532 and miR-93 were proportionally ⩾8-fold more abundant in the nucleolus than in the other respective compartments (Fig. 5B). These findings suggested that several miRNAs showed compartment-specific enrichment.

3.4 Dicer inactivation does not affect miRNA nucleolar accumulation

Dicer has been suggested to affect processing of certain snoRNAs to miRNA-sized fragments [40, 41]. To address whether Dicer affects miRNA nucleolar presence, we analyzed small RNAs from Dicer−/− HCT116 cells and the parent Dicer-proficient HCT116 cells. It has been previously shown that miR-21 processing is compromised in the Dicer−/− HCT116 cells, whereas miR-31 processing to mature form is less affected [46]. We hence used subcellular RNA fractions of the HCT116 parent and Dicer-null derivative cells to analyze for miR-21 and miR-31 by Northern blotting. As shown in Supplementary Fig. S2, miR-21 was proportionally decreased in the Dicer-null cells in all cellular compartments. Similarly, the decrease in abundance of miR-31 was proportional. Hence, ablation of Dicer did not alter the nucleolar presence of miR-21.

3.5 MiR-21 and miR-31 nucleolar association increases following CRM1 inhibition

As shown by numerous studies, Pol I transcription blocks cause nucleolar disintegration and reorganization of the nucleolar proteome and RNome [28, 39, 47, 48]. We therefore used Actinomycin D (ActD) to inhibit Pol I to ask whether nucleolar miRNA abundance would be affected. Interestingly, although the presence of hY1 was increased in the nucleolar fraction of ActD-treated cells, that of miR-31 was unaffected (Fig. 6 A). This suggests that ongoing Pol I transcription may not be required for the nucleolar presence of miRNAs.

CRM1/NMD3 export/adaptor complex extensively accumulates in the nucleolus when Pol I is inhibited [35, 39]. This is especially interesting as CRM1 is involved in the nucleolar trafficking of snoRNA [33], and has been implicated in the trafficking of miRNAs [49]. We treated the cells with a CRM1-specific inhibitor, leptomycin B. Subcellular fractionation and analysis of miR-31 by Northern hybridization indicated that the nucleolar abundance of miR-31 was increased by 3-fold in leptomycin B-treated cells (Fig. 6B and C).