Materials and Methods

A small RNA is linking CRISPR-Cas and zinc transport

Strains and Growth conditions

Strains and oligonucleotides used in this study are listed in Supplementary Table 3 and 4. A detailed description of the media used can be found in the Supplementary Data. Cloning procedures were performed using E. coli strain DH5α and standard culture (aerobically, 37°C, 2YT media) as well as molecular biological techniques (Sambrook, 2006). H. volcanii strains were grown aerobically at 45°C in either YPC, Hv-Ca, Hv-MM with appropriate supplements (Allers et al., 2004; Duggin et al., 2015; de Silva et al., 2020). For in-depth comparison of growth, transcriptome and proteome, H66 (Δpyr_E2, Δ_leu_B) was used as wild type, since in the s479 deletion strain, the s479 gene is replaced by a tryptophan marker in the genome of the parent strain H119 (Δ_pyr_E2, Δ_trp_A, and Δ_leu_B). Thus Δ_s479 and H66 require both addition of uracil and leucine to media for growth (Allers et al., 2004; Jaschinski et al., 2014).

Growth Experiments

Growth experiments were carried out in microtiter plates using a heated plate reader (Epoch 2 NS Microplate Spectrophotometer, BioTek Instruments). Strains H66 (wild type) and Δs479 were precultured in Hv-Ca medium supplemented with uracil to OD650nm= 0.4-0.7 and then diluted to OD650nm=0.05 and transferred to microtiter plates in triplicates. These were then cultured (aerobically, orbital shaking, 45 °C) while OD650nm was measured every 30 min. Outer wells were filled with salt water as evaporation barriers (Jaschinski et al., 2014; Liao et al., 2016). For stress conditions, adjusted media preparations were used (see above "Strains and Growth conditions"). Doubling time (d h) and growth rate (μ h−1) were calculated as growth rate μ = (ln(xt) – ln (x0)) / (t – t0) and doubling time d= ln(2) / μ. Calculations were carried out separately for all replicates before calculating mean value and standard deviation. Phases of exponential growth were identified using fitted trendlines and corresponding R2-values (Supplementary Figure 5B. and 5C.)

Northern Blot Analysis

For analysis of s479 transcripts (Figure 2) Haloferax strains H119 and Δs479 were cultivated in Hv-MM supplemented with leucin and uracil (for H119 tryptophan was also added); for detection of znu mRNAs (Figure 6) Haloferax strains H66 and Δs479 were grown in YPC. For detection of s479 transcripts in Figure 10 Haloferax strain H119 was cultivated in Hv-MM supplemented with leucin, tryptophan and uracil; deletion strains Δcas6 and Δcas7 were cultivated in YPC. For investigation of znu mRNAs in Cas protein deletion strains Δcas6 and Δcas7 (Supplementary Figure 4) H119, Δcas6 and Δcas7 were grown in YPC. TRIzol™ Reagent (Invitrogen™, ThermoFisher Scientific) or NucleoZOL™ (Machery and Nagel) was used to isolate total RNA from H. volcanii cells. Ten microgram total RNA was separated using an 1.5 % agarose (transcript size >500 nt) or 8 % denaturing polyacrylamide gel (PAGE) and then transferred to a nylon membrane (Biodyne® A, PALL or Hybond-N+, GE Healthcare). After transfer, the membrane of PAGE blots was hybridized with oligonucleotide s479spacerpart (primer sequences are listed in Supplementary Table 4) to detect the s479 transcript, the membrane was subsequently hybridised with an oligonucleotide against the 5S rRNA, both radioactively labelled with γ-32P-ATP via polynucleotide kinase treatment. Membranes of agarose blots were hybridized with a probe against znu_C1 generated by PCR using primers "probe znuC1 Hvo_2398 fw/rev" and genomic DNA as template and the product was labelled using α-32P-dCTP and the random primed DNA labelling kit DECAprime™II (Invitrogen). In addition, the membrane was hybridised with a probe against the 16S rRNA. The probe was generated with PCR using primers 16Sseqf and 16Sseqrev and genomic DNA from _H. volcanii as template. Using the DECAprime™II kit (Invitrogen) the PCR fragment was radioactively labelled with α-32P-dCTP. Oligonucleotide probes and PCR primers see Supplementary Table 4.

Sample Preparation for transcriptome analysis and RNA-seq analysis

Three replicates of wild type (H66) and deletion strain (Δs479) were cultured in Hv-Ca medium supplemented with uracil at 45°C and grown to OD650nm=0.6-0.7. Total RNA was isolated using NucleoZOL™ (Machery and Nagel) and RNA samples were sent to vertis Biotechnologie AG (Martinsried, Germany) for further treatment. Total RNA was treated with T4 Polynucleotide Kinase (NEB) and rRNA depleted using an in-house protocol and cDNA library preparation was preceded by ultrasonic fragmentation. After 3’ adapter ligation, first-strand cDNA synthesis was performed using 3’-adapter primer and M-MLV reverse transcriptase. After cDNA purification, the 5’ Illumina TruSeq sequencing adapter was ligated to the 3’ end of the antisense cDNA and the sample amplified to 10-20 ng/μl using a high-fidelity DNA polymerase. Finally, cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics), samples were pooled (equimolar), the pool size fractionated (200 – 550 bp) by preparative agarose gel electrophoresis and sequenced on an Illumina NextSeq 500 system using 1×75 bp read length. TruSeq barcode sequences which are part of the 5’ TruSeq sequencing adapter are included in Supplementary Table 4. Sequencing reads are deposited at the European Nucleotide Archive (ENA) under the study accession number PRJEB41379. For data analysis, reads were mapped to the genome using bowtie2 (version2.3.4.1) with the „--very-sensitive” option and defaults otherwise (Langmead et al., 2009). Then reads per feature were counted using featureCounts (version 1.6.4) and analysed for differential expression with DeSeq2 (version 1.2.11) (Liao et al., 2014; Love et al., 2014).

Sample Preparation for MS/MS Analysis

Three biological replicates (250 ml) of wild type (H66) and deletion strain (Δs479) were cultivated in Hv-Ca media supplemented with uracil, 45°C and grown to OD650nm= 0.6-0.74. Cells were harvested and washed in enriched PBS buffer (2.5 M NaCl, 150 mM MgCl2, 1× PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 2 mM K2HPO4, pH 7.4). After cell lysis by ultrasonication in 10 ml lysis buffer (1 M NaCl, 100 mM Tris/HCl, pH 7.5, 1 mM EDTA, 10 mM MgCl2, 1 mM CaCl2, 13 μl/ml protease inhibitor mixture (Sigma)) cytosolic and membrane fraction were separated by ultracentrifugation at 100.000 x g and treated as separate samples. The cytosolic protein sample was directly used for 1D SDS PAGE whereas the pelleted membrane protein fraction was solubilized in 2 ml HTH buffer ((6 M Thiourea/2 M Urea); 10 min 37°C; 10 min 37°C ultrasonication). 20 μg of both samples were separated by 1D SDS PAGE and in gel digested as previously described (Bonn et al., 2014). Briefly, Coomassie stained gel lanes were cut resulting in ten gel pieces per sample before gel pieces were cut into smaller blocks and transferred into low binding tubes. Samples were destained and dried in a vacuum centrifuge before being covered with trypsin solution. Digestion was carried out at 37 °C overnight before peptides were eluted in water by ultrasonication. The peptide-containing supernatant was transferred into a fresh tube, desiccated in a vacuum centrifuge and peptides were resolubilized in 0.1% (v/v) acetic acid for mass spectrometric analysis.

MS/MS Analysis

LC-MS/MS analyses were performed on an LTQ Orbitrap Velos Pro (ThermoFisher Scientific, Waltham, Massachusetts, USA) using an EASY-nLC II liquid chromatography system. Tryptic peptides were subjected to liquid chromatography (LC) separation and electrospray ionization-based mass spectrometry (MS) applying the same injected volumes in order to allow for label-free relative protein quantification. Therefore, peptides were loaded on a self-packed analytical column (OD 360 μm, ID 100 μm, length 20 cm) filled with 3 μm diameter C18 particles (Dr. Maisch, Ammerbuch-Entringen, Germany) and eluted by a binary nonlinear gradient of 5 - 99 % acetonitrile in 0.1 % acetic acid over 86 min with a flow rate of 300 nL/min. For MS analysis, a full scan in the Orbitrap with a resolution of 30,000 was followed by collision-induced dissociation (CID) of the twenty most abundant precursor ions. MS2 experiments were acquired in the linear ion trap.

MS Data Analysis

Database search was performed with MaxQuant 1.6.17.0 against a H. volcanii database (Jevtić et al., 2019) containing 4106 entries. Max Quant’s generic contamination list as well as reverse entries were added during the search. The following parameters were applied: digestion mode, trypsin/P with up to 2 missed cleavages; variable modification, methionine oxidation and N-terminal acetylation, and maximal number of 5 modifications per peptide; activated LFQ option with minimal ratio count of 2 and ‘match-between runs’ feature. The false discovery rates of peptide spectrum match and protein level were set to 0.01. A protein was considered to be identified if two or more unique peptides were identified in a biological replicate. Only unique peptides were used for protein quantification.

The comparative proteome analyses based on MaxQuant LFQ values were performed separately for cytosolic and membrane protein samples. Proteins were considered to be quantified if a quantitative value based on at least two unique peptides was available in at least two biological replicates. LFQ values as proxy for protein abundance were used for statistical analysis. A Student’s t-tests was performed to analyse changes in protein amounts between wild type and mutant. Proteins with significantly changed amount exhibited a p-value < 0.01 and an average log2 fold change >|0.8|

Electrophoretic mobility shift assay

For electrophoretic mobility shift assays (EMSA) RNAs were obtained from Biomers (Biomers, Ulm, Germany) (sequences are listed in Supplementary Table 4). The s479 RNA was labelled at the 3’ end using α-32PpCp and T4 RNA ligase (Fermentas, Thermo Fisher Scientific). For the EMSA in Figure 9, 100 cps labelled s479-RNA was mixed with 50, 100 or 200 pmol the unlabelled _znu_C1 RNA fragment encompassing the interaction site 2 (Figure 7D.). For the EMSA in Supplementary Figure 3A., 100 cps labelled s479 RNA was mixed with 50 pmol unlabelled _znu_C1 RNA encompassing the interaction site 2 (Figure 7D.), in addition 0, 50, 200 or 400 pmol of unlabelled s479 were added. For the EMSA in Supplementary Figure 3B., 100 cps labelled s479 RNA was mixed with 50 pmol unlabelled _znu_C1 RNA encompassing the interaction site 2 (Figure 7D.) or 50, 200 or 400 pmol of unlabelled _znu_C1 RNA mutant, which has the s479 interaction site deleted (Figure 7D.) All reactions were performed in 20 μl reaction volume containing 10 mM Tris-Cl pH 7.5, 5 mM MgCl2 and 100 mM KCl. After incubation at 37°C for 30 min 1 μl 50% (vol/vol) glycerol containing 0.1% (w/vol) bromphenol blue was added and samples separated on a native 8% (w/vol) polyacrylamide gel at 4°C which was subsequently analysed by autoradiography.

In silico target site prediction

To predict target sites of s479 in silico, we applied IntaRNA (version 3.2.0) (Mann et al., 2017). For prediction of potential s479 interaction sites, we used the s479 sequence corresponding to pHV4: 207,716-770. This corresponds to the start point of the potential spacer sequence of the shortened s479 versions (Figure 3). The spacer is the sequence located downstream of the 5’ handle sequence within crRNAs. In crRNAs, the spacer sequence is the sequence used for target recognition. Therefore we chose this part of the sequence for analysis and set the spacer length to 55 nt. IntaRNA was used with default settings for prediction of the s479::_znu_C1 interaction sites. For prediction of the interaction sites on the proteome-targets, we first used default settings and then also included predictions for a seed sequence of five nucleotides as this is the increment of protein contacts seen for spacer sequences within Cascade complexes (Maier et al., 2019).

Article TitleA small RNA is linking CRISPR-Cas and zinc transport

Abstract

The function and mode of action of small regulatory RNAs is currently still understudied in archaea. In the halophilic archaeon H. volcanii a plethora of sRNAs have been identified, however, in-depth functional analysis is missing for most of them. We selected a small RNA (s479) from H. volcanii for detailed characterization. The sRNA gene is encoded between a CRISPR RNA locus and the Cas protein gene cluster, the s479 deletion strain is viable and was characterized in detail. Transcriptome studies of wild type Haloferax cells and the deletion mutant revealed up-regulation of six genes in the deletion strain, showing that the sRNA has a clearly defined function. Three of the six up-regulated genes encode potential zinc transporter proteins (ZnuA1, ZnuB1, ZnuC1) suggesting involvement of s479 in regulation of zinc transport. Upregulation of these genes in the deletion strain was confirmed by northern blot and proteome analyses. Furthermore, electrophoretic mobility shift assays demonstrate a direct interaction of s479 with the target znu_C1 mRNA. Proteome comparison of wild type and deletion strains further expanded the regulon of s479 deeply rooting this sRNA within the metabolism of _H. volcanii especially the regulation of transporter abundance. Interestingly, s479 is not only encoded next to CRISPR-cas genes but the mature s479 contains a crRNA-like 5’ handle and experiments with Cas protein deletion strains indicate maturation by Cas6 and interaction with Cas proteins. Together this might suggest that the CRISPR-Cas system is involved in s479 function.


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