Separation of cell types
About 5000 asexual growing V. carteri spheroids at the stage shortly before the onset of embryogenesis were collected by filtration on a 100 μm mesh nylon screen and broken by passing them through a 0.6 mm hypodermic needle. The suspension was filtered on a 100 μm mesh nylon screen, which allows free reproductive cells (gonidia), free somatic cells and small fragments of the somatic cell layer to pass but retaining larger fragments of somatic cell layers nearly free from gonidia. The filtrate was passed in a second step through a 40 μm mesh nylon screen which retained only gonidia and small fragments of the somatic cell layer. The resuspended residue was allowed to settle down repeatedly in a small volume separating the reproductive cells from somatic cell layers.
The separation of gonidia and somatic cell layers of sexual induced V. carteri spheroids at the stage shortly before the onset of embryogenesis and 16 h after application of the sex-inducing pheromone was done in the same manner as described above.
The separation of egg cells and somatic cells of sexual growing V. carteri spheroids 64 h after application of the sex-inducing pheromone was done in a modified procedure: Egg cells were set free by passing the spheroids twice through a 0.5 mm hypodermic needle. A first filtration step on a 40 μm mesh nylon screen retained large fragments of the somatic cell layer. Egg cells which pass through were collected on a 10 μm mesh nylon screen and purified further by let them settle down repeatedly in a small volume. The residue of the first filtration step was dissociated once more by passing through a 0.4 mm hypodermic needle. Fragments of the somatic cell layer were collected on a 40 μm mesh nylon screen.
Production of stable V. carteri strains using the gold particle gun
Precipitation of DNA (plasmids) onto gold-microcarriers
For each transformation, 10 μg of plasmid DNA encoding the selection marker (pPmr3, see [29]) and 10 μg plasmid encoding the target gene were used. The DNA solution was added to 0.5 μmol gold particles while mixing vigorously. Continuing the shaking, 125 μmol CaCl2 (50 μl of 2.5 M) and 2 μmol spermidin (20 μl of 100 mM) were added. The mixture was further incubated at 4 °C for 30 min under continuous shaking. To precipitate the DNA onto the gold, 200 μl of 100 % ethanol were added, followed by a short centrifugation step (3–4 s at 8000 rpm). The gold particles were washed three times with ice cold 100 % ethanol and taken up in a total volume of 40 μl. The particles were stored on ice until transformation.
Nuclear transformation
Nuclear transformation was carried out according to Jakobiak et al., 2004 [29]. One aliquot of gold particles were used to transform V. carteri spheroids from one Fernbach flask. For this, the gold particle mixture was spread over the center of 6 macrocarriers (fixed in their metal carriers). The macrocarriers were warmed on a heating block at 37 °C to evaporate residual ethanol. One macrocarrier, stopping screen and rupture disk (900 psi) were placed into the apparatus. The V. carteri spheroids were harvested using a sieve and spread in its center. The sieve was placed directly below the macrocarriers. Transformation was carried out under vacuum using the biolistic PDS 1000/He particle gun (BioRad Laboratories, Hercules, USA). For one transformation, the V. carteri spheroids were six times spread on the sieve, bombarded with gold particles and submerged in V. carteri medium during change of the carriers and disks.
Following bombardment, the spheroids were split into ten petri dishes containing 30 ml medium. Two days after transformation, 30 μg/ml paromomycin (selection marker) was added to each plate. After selection of paromomycin positive clones, the concentration was decreased to 10 μg/ml.
Cloning and sequencing of RNA transcripts
The RNA Seq libraries were generated from 4 μg total RNA per sample. The TruSeq Sample Preparation Kit (Illumina) was used according to the manufacturer’s instructions. The only derivation of the protocol was the substitution of the Superscript II reverse transcriptase with the Superscript III reverse transcriptase (both Life Technologies). Accordingly, the cDNA synthesis temperature was raised from 42 °C to 50 °C. Libraries of two biological replicates were generated on different days and sequenced on a HiSeq 2000 at the facilities of Illumina in a 100 bp paired end run. Additionally, the same libraries were sequenced in single runs (100 bp) with GATC (Konstanz, Germany). For the generation of strand-specific libraries, samples of all cells in all life stages were pooled. Total RNA was enriched for mRNA using the polyA Purist Kit (Life Technologies Cat No AM1919). Library was made with SOLiD total RNA-seq kit (Life Technologies Cat No 4445374) in accordance with manufacturer’s protocol and sequence on SOLiD 3 platform.
Northern blotting
Northern blotting was performed as described before [30]. In short, 5–10 μg of total RNA were run on a 12 % urea gel (UreaGel System, National diagnostics). For determining the approximate size of the small RNAs, ribooligonucleotides with a length of 19, 21 and 24 nucleotides (nt) were labeled with 32P prior to loading. The gel was run for 1 h at 250–350 V and semi-dry blotted onto an Amersham Hybond-N membrane (GE Healthcare) at 20 V for 30 min. Cross-linking with EDC-solution was performed at 50 °C for 1 h. The membrane was subsequently rinsed in water, dried and incubated with hybridization solution at 50 °C. After pre-hybridization, a radiolabeled probe antisense to the target small RNA was added and incubated over night at 50 °C. For labeling, 20 pmol of the probe (DNA oligonucleotide) were incubated with 20 μCi of 32P in a T4 PNK reaction (Fermentas) and it was purified with a G-25 column (GE Healthcare). After the incubation, the membrane was washed twice with 5xSSC, 1 % SDS, once with 1xSSC, 1 % SDS and wrapped in saran. The detection of signals was performed by the exposure to a screen and scanning with the PMI (Biorad).
For the re-probing of a membrane, the membrane was incubated with hot water with 0.1 % SDS for at least 15 min on a tumbler. The membrane was exposed to a screen for at least overnight to control for residual signal.
For the assessment of band heights when the radioactive marker had faded, the distance between the blue dye band heights was measured (marked with pencil on the membrane before blotting) as well as the distances of the marker bands to the blue dye bands. These figures were used to estimate band heights in later blots.
Data analysis
Analysis of RNA-Seq data
All small RNA sequencing data were adapter- and quality score-trimmed (trailing/leading base Phred score > 20). Resulting reads were aligned to the V. carteri reference genome version 9.0 [31] using tophat2/bowtie2 [32].
Paired-end total RNA sequencing data were also adapter- and quality score-trimmed (trailing/leading base Phred score > 30), and aligned to the same V. carteri reference genome version 9.0.
Transcriptome assembly
Paired-end and single-end reads from libraries V1-V12 were mapped each separately to the V. carteri genome assembly vc199 (Phytozome v.8 [31]) using TopHat software v.1.4.1 [33] and separate de novo transcriptome assemblies were generated for each library using Cufflinks software V 2.0.1 [33] with default parameters. Next, all assemblies were merged using cuffmerge program from the Cufflinks package. Finally, transcript orientation was corrected by mapping strand-specific SOLiD RNA-seq data on the transcripts using Bowtie software v.0.12.7 [32] and assigning particular orientation if the number of reads mapping to one strand was at least two times greater than the number of reads mapping to the opposite strand. In cases where this was not possible, no strand correction was performed.
Identification of transcripts with a putative role in RNAi
HHblits [34], which is part of the HHsuite, was used to search for fragments of RNAi processing proteins in the transcriptome of V. carteri. We used the current transcript assembly v.2 of the JGI (Joint Genome Institute), which is freely available at the Phytozome 10 database [31]. In addition, the transcriptome data generated by us was used for this analysis (see paragraph above). HHblits requires for each protein to be searched for a hidden-Markov-model (HMM) to be generated by the user. These HMMs constitute a custom database, which was compiled according to the protocol detailed in chapter 3.4 of the HHSuite User Guide (downloaded from https://toolkit.tuebingen.mpg.de). To initiate the compilation of the respective HHMs, the following proteins from Arabidopsis thaliana were used as a seed: DCL4 (UniProtKB ID P84634), DCL1 (Q9SP32), HEN1 (Q9C5Q8) and RdRP6 (Q9SG02). For the search of additional Argonaute genes, the modified V. carteri AGO3 was used. For each sequence, a multiple sequence alignment was created by using HHblits applied to the database uniprot20_2013_03 from the EBI (http://www.uniprot.org/). As required, secondary structure was predicted by means of psipred_3.5 [35] and added to the HHblits alignments. The compilation of the database was finalized according to the above-mentioned protocol. Since the open reading frames of annotated V. carteri proteins were not always supporting full-length proteins with start and stop codons, different protein sequences were created. DNA was translated in the six putative reading frames to protein sequences by means of methods from the Biophython package (https://github.com/biopython/biopython.github.io/). HHblits hits with an E-value ≤ 10E−5 were considered important and further processed. Next, the genomic locus of each transcript was extracted and the list of transcripts was sorted accordingly. Transcripts were grouped according to coverage of the query protein, e.g. for Argonaute, two transcripts in close proximity with one encoding the N-terminal and the other encoding the C-terminal part were considered to be an important hit. To further validate the candidate genes, the protein search algorithm of Pfam version 27.0 [36] and Panther HMM Sequence Scoring [37] were employed to identify single important domains in the respective transcripts.
miRNA identification
The miRNA identification was performed using the novel miRNA identification tool miRA [19]. miRA does not assume sequence conservation, and was developed to characterize the miRNA landscape in organisms exhibiting heterogeneous miRNA precursor populations. Key parameters were chosen based on an analysis of miRBase-annotated miRNAs in C. reinhardtii. Details involving the identification method can be found in Evers et al. [19].
Annotation of other small RNAs
Reads overlapping annotated exons of mRNA transcripts were assigned to the mRNA fraction. Gene annotations were based on the V. carteri reference genome version 9.0 [31]. Since there are no databases listing V. carteri rRNA and tRNA genes in full, reads were mapped against the database entries for C. reinhardtii tRNAs (PlantRNA database, [38] and rRNAs from A. thaliana (SILVA database, [39] and the order Volvocales (exported from GenBank, NCBI). Repeats were assigned using Repbase Update 19.02 [40], phased RNAs were predicted using the ta-si prediction tool from the UEA small RNA Workbench [41] which is based on the algorithm by Chen et al. [42].
miRNA target prediction
Potential miRNA targets in V. carteri were identified by requiring the following set of matching rules between the miRNA seed region and complementary mRNA binding site (adapted from [14]): (a) the binding site of the miRNA should have no more than four mismatches, (b) there should not be a mismatch at positions 10 an 11 of the miRNA, because complementarity here is required for cleavage, (c) there should not be adjacent mismatches at positions 2–12, (d) no more than two adjacent mismatches for positions > 12 are allowed and (e) there should be no bulge in the miRNA.
Mapping of small RNAs to repetitive elements
To investigate transcription of small RNAs from repetitive elements, the following strategy was employed: Small RNA reads were mapped to a list of RepBase-derived V. carteri repeat elements using tophat2/bowtie2. Overall the AgoIP sequencing library showed the strongest repeat element-associated expression, with Kangaroo, Gypsy3–5, and Jordan being amongst the most highly expressed repeat elements in the sample.
Generation of logos
All sequence logos were based on the adapter- and quality score-trimmed reads, and were generated using the R package motifStack (Ou and Zhu, R package version 1.12.0).
Generation of multiple sequence alignment
All sequences of miRNAs of V. carteri and C. reinhardtii (miRBase) were subjected to a multiple sequence alignment using ClustalW [43]. The resulting matrix of alignment quality scores was plotted as a heat map of the pairwise score as percentage identity. The same analysis was performed with miRNAs from the liverwort Pellia endiviifolia [26].
Strains and culture conditions of V. carteri and C. reinhardtii
The female Volvox carteri f. nagariensis wild-type strain HK10 was originally obtained from R.C. Starr (Culture Collection of Algae, University of Texas, Austin, Texas, USA). The strain Vol6♀ was obtained from A. Hallmann (University of Bielefeld, Germany). Synchronous cultures were grown in Volvox medium at 28 °C under an 8 h dark/16 h light (10 000 lux) cycle [3]. The sex-inducing pheromone was used as described by Haas and Sumper [44].
The cell-wall deficient strain CW15 mt- was obtained from Jörg Nickelsen (University of Munich) and cultivated on agar plates from TAP medium at room temperature on a shelf exposed to natural sunlight. TAP medium and its ingredients were made as described at chlamy.org, originally described in Gorman et al. [45]. Liquid cultures were grown in TAP medium in Erlenmeyer flasks at room temperature and exposed to natural sunlight.
Immunoprecipitation and protein blots
Approximately 1 ml (myc-VcAGO3 transformed) or 400 μl (myc-CrGFP transformed) of V. carteri spheroids were lysed in 5 ml or 2 ml of lysis buffer (150 mM KCl, 25 mM Tris/HCl pH7.4, 0.5 % NP-40, 2 mM EDTA, 1 mM NaF), respectively, and sonicated twice for 30s (cycle 5, 10 % intensity, MS72 tip, Bandelin Sonoplus). After centrifugation at 17000 g, 4 °C and 30 min, the supernatant was flash frozen in liquid nitrogen. After thawing on ice the next day, the lysates were incubated with anti-c-myc-beads (Sigma) and incubated for 2.5 h at 4 °C on a rotating wheel. Beads were washed three times with wash buffer (300 mM KCl, 50 mM Tris/HCl pH7.4, 1 mM MgCl2, 0.1 % NP-40) and once with PBS.
For protein blots, 10 μl of the respective lysate as input and 20 % of the immunoprecipitation (IP) of the myc-VcAGO3 transformation and all of the myc-CrGFP transformation was used. For the control of immunoprecipitation of the cleavage assays, 10 % of each reaction was used.
Standard protein blots were performed using a 10 % separating gel. Semi-dry blotting using Towbin buffer was performed with 1.5 mA/cm2 for 2 h on Hybond ECL membrane (GE Healthcare). After blocking for 30 min with 5 % milk powder in TBS-T (TBS with 0.2 % Tween20), the membrane was incubated with the primary antibody (anti-myc, Sigma, 1:1500; anti-Flag 1:1000) over night at 4 °C. After washing with TBS-T, the membrane was incubated with the secondary antibody (anti-rabbit IRDye 800CW, Licor, 1:10000) for 1 h at room temperature. After washing, the membrane was scanned on a Licor Odyssey reader.
RNA preparations
For the extraction of RNA from somatic cells or reproductive cells, 3 ml peqGold Trifast (Peqlab) or Trizol (Life technologies) was added per 100 μl cell suspension. Cells were lysed at room temperature for 10 min and RNA preparation was then carried out according to the manufacturer’s instructions. Precipitation of the RNA was performed over night at −20 °C in the presence of 20 μg glycogen (RNA grade, Life technologies) per ml of Trifast/Trizol. The RNA was collected by centrifugation, washed once with cold 80 % ethanol and solved in pure water.
RNA from immunoprecipitation samples was prepared as described [30].
Cleavage assay
Before assessing the cleavage activity, AGO proteins and the negative control CrGFP were precipitated via their respective tags. For VcAGO3 and CrGFP, the IP was carried out with 0.5 ml pellets as described under section “Immunoprecipitation and protein blots”. For lysing Arabidopsis tissue, 250 mg of tissue powder were incubated with 1 ml of ice cold extraction buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 10 % Glycerol, 5 mM MgCl2, 0.1 % NP-40, 5 mM DTT), mixed well and incubated on a rotating wheel at 4 °C for 1 h. After clearing the cell debris by centrifugation (17,000 g, 4 °C, 30 min), 1 ml of lysate was added to 30 μl of packed Flag-beads (anti-Flag M2, Sigma) and incubated for 2 h at 4 °C on a rotating wheel. After three washes with wash buffer (50 mM Tris HCl pH 7.5, 500 mM NaCl, 10 % Glycerol, 5 mM MgCl2, 0.1 % NP-40, 4 mM DTT), PBS was used to split the immunoprecipitated AtAGO1 into fresh tubes. For checking the success of immunoprecipitation, 10 % of each IP (Volvox and Arabidopsis) were used for Western blotting.
The preparation of 32P-cap-labeled target RNA was performed as described before [8]. For VcAGO3 cleavage assays, targets were generated carrying a perfectly complementary site to the small RNAs tasiRNA#1, MIR357b and sRNA scaff 12, while target RNAs for AtAGO1 cleavage assays carried sites for MIR159a and MIR165. The in vitro cleavage reaction was carried out with 50 % (v/v) immunoprecipitated protein (beads) in 66.7 mM KCl, 6.7 mM MgCl2, 8.3 mM DTT, 1.7 mM ATP, 0.3 mM GTP and 3.2 U RiboLock RNase inhibitor (Thermo Scientific). The addition of target RNA (1–2 Bq/cm2) initiated the reaction which was carried out for 1.5 h at 25 °C. The RNA was subsequently extracted using Proteinase K digestion and phenol-chloroform extraction as described before [46].
To visualize the cleavage products, samples were run on an 8 % urea polyacrylamide sequencing gel (National diagnostics), dried on Whatman paper and exposed to a screen.
Cloning and sequencing of small RNAs
Small RNAs from total RNA samples were converted into sequencing libraries as described before [30]. The libraries were sequenced by Fasteris SA (Geneva, Switzerland) on an Illumina HiSeq2000 in a 50 bp single-end run. The RNA that was extracted from a VcAGO3 immunoprecipitation was cloned as described before [47] and sequenced on a MiSeq (Illumina) in a 66 bp single-end run.
β-elimination treatment of small RNAs
Total RNA from vegetative somatic cells and vegetative gonidia was mixed with 20 pmol of a random oligo RNA (5′-UUAGUGAGAGUCCAAUUAAUU-3′, Biomers). Beta-elimination was performed as described previously [48].
13.5 μl of total RNA (10–20 μg, vegetative somatic cells or vegetative gonidia) were mixed with 4.5 μl of 5x borate buffer (148 mM borax, 148 mM boric acid, pH 8.6) and 2.5 μl of freshly dissolved 200 mM NaIO4. After incubation for 10 min at room temperature, 2 μl of glycerol were added to quench unreacted NaIO4. Samples were incubated for another 10 min at room temperature and then dried by vacuum centrifugation for 1 h at room temperature.
Samples were dissolved in 50 μl 1x borax buffer (30 mM borax, 30 mM boric acid, 50 mM NaOH, pH 9.5) and incubated at 45 °C for 90 min. 20 μg glycogen was added to each sample and the RNA was precipitated with 2.5 volumes of ethanol at −20 °C over night. RNA was collected by centrifugation at 17000 g, 4 °C, 30 min. Pellets were directly dissolved in RNA loading dye.