ena-STUDY-GARVAN INSTITUTE-29-03-2019-03:40:48:441-176
The SRPome reveals SRP partners and non canonical functions
The signal recognition particle (SRP) is a ribonucleoprotein complex that assembles in a spatially and temporally regulated manner. Although SRP plays an essential role in co-translational targeting of proteins, new functions beyond the SRP cycle have started to emerge, including gene expression, apoptosis and stress response. This study presents the first comprehensive analysis of the eukaryotic SRP-binding partners. Two layers of the SRP interactome (SRPome), the SRP proteome and SRP transcriptome, were analysed by LC-MS/MS and RIP-seq, respectively. The majority of the newly identified protein and RNA partners of the SRP are involved in ribonucleoprotein particles (RNPs) formation, RNA processing and protein transport. SRP components have a dynamic cellular localization during the cell cycle. SRP proteins were shown to interact and co-localize with PRMT1 and PRMT5, while SRP54 was also shown to co-localize with the paraspeckle component NONO. Together these data provide a rich resource for identifying novel SRP partners and reveal several noncanonical functions of SRP that are previously unknown.  Sample preparation: NTERA-2 cells were grown in DMEM media (Gibco) to 80% confluence in 15 cm dishes and crosslinked with 1% (v/v) formaldehyde for 10 minutes at RT. The cross-linking reaction was quenched by incubating for 5 minutes with glycine to a final concentration of 125 mM, followed by two washing steps with ice-cold PBS. Lysates were clarified from sonicated nuclei and SRP-RNA complexes were isolated with antibody. Libraries were prepared according to Diagenode's instructions, using CATS-RNA seq kit C05010041. Briefly, 10 ng of single stranded RNA were first chemically fragmented at 94 °C for 2 minutes, end-repaired and polyadenylated at the 3' end. Subsequently, a cDNA strand synthesis was performed in the presence of the anchored poly(dT) oligonucleotides containing terminal P7 Illumina adaptor sequence.  During PCR pre-amplification (10 cycles) of the first cDNA strand, Illumina adapters carrying P5 and P7 terminal sequences as well as index sequences are incorporated into the library. The library was clean up twice with 0.9X AMPure XP beads (Beckman Coulter Life Sciences) to remove small DNA fragments and eluted in 20 µl of 1X TE buffer.  Analysis: Trimming - 1 stage Trimmomatic 0.36 Reads were trimmed with Trimmomatic 0.36 with parameters (ILLUMINACLIP:TruSeq3-PE.fa:2:30:5 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:18 HEADCROP:8)  Trimming - 2 stage Cutadapt 1.18 Adapters and polyA sequences were removed by Cutadapt 1.18 Alignement  STAR 2.4.0.1 Reads were aligned to hg38 genome using a 2-pass mode basic procedure Peak-calling  RIPSeeker 3.8
The signal recognition particle (SRP) is a ribonucleoprotein complex that assembles in a spatially and temporally regulated manner. Although SRP plays an essential role in co-translational targeting of proteins, new functions beyond the SRP cycle have started to emerge, including gene expression, apoptosis and stress response. This study presents the first comprehensive analysis of the eukaryotic SRP-binding partners. Two layers of the SRP interactome (SRPome), the SRP proteome and SRP transcriptome, were analysed by LC-MS/MS and RIP-seq, respectively. The majority of the newly identified protein and RNA partners of the SRP are involved in ribonucleoprotein particles (RNPs) formation, RNA processing and protein transport. SRP components have a dynamic cellular localization during the cell cycle. SRP proteins were shown to interact and co-localize with PRMT1 and PRMT5, while SRP54 was also shown to co-localize with the paraspeckle component NONO. Together these data provide a rich resource for identifying novel SRP partners and reveal several noncanonical functions of SRP that are previously unknown.  Sample preparation: NTERA-2 cells were grown in DMEM media (Gibco) to 80% confluence in 15 cm dishes and crosslinked with 1% (v/v) formaldehyde for 10 minutes at RT. The cross-linking reaction was quenched by incubating for 5 minutes with glycine to a final concentration of 125 mM, followed by two washing steps with ice-cold PBS. Lysates were clarified from sonicated nuclei and SRP-RNA complexes were isolated with antibody. Libraries were prepared according to Diagenode's instructions, using CATS-RNA seq kit C05010041. Briefly, 10 ng of single stranded RNA were first chemically fragmented at 94 °C for 2 minutes, end-repaired and polyadenylated at the 3' end. Subsequently, a cDNA strand synthesis was performed in the presence of the anchored poly(dT) oligonucleotides containing terminal P7 Illumina adaptor sequence.  During PCR pre-amplification (10 cycles) of the first cDNA strand, Illumina adapters carrying P5 and P7 terminal sequences as well as index sequences are incorporated into the library. The library was clean up twice with 0.9X AMPure XP beads (Beckman Coulter Life Sciences) to remove small DNA fragments and eluted in 20 µl of 1X TE buffer.  Analysis: Trimming - 1 stage Trimmomatic 0.36 Reads were trimmed with Trimmomatic 0.36 with parameters (ILLUMINACLIP:TruSeq3-PE.fa:2:30:5 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:18 HEADCROP:8)  Trimming - 2 stage Cutadapt 1.18 Adapters and polyA sequences were removed by Cutadapt 1.18 Alignement  STAR 2.4.0.1 Reads were aligned to hg38 genome using a 2-pass mode basic procedure Peak-calling  RIPSeeker 3.8