Noncanonical small RNA chimeras identified by AGO2-CLASH

a 2022

Noncanonical small RNA chimeras identified by AGO2-CLASH

HEJRET, Václav, Nandan Mysore VARADARAJAN, Eva KLIMENTOVÁ, Ilektra-Chara GIASSA, Štěpánka VAŇÁČOVÁ et. al.

Základní údaje

Originální název

Noncanonical small RNA chimeras identified by AGO2-CLASH

Autoři

HEJRET, Václav, Nandan Mysore VARADARAJAN, Eva KLIMENTOVÁ, Ilektra-Chara GIASSA, Štěpánka VAŇÁČOVÁ a Panagiotis ALEXIOU

Vydání

16th Microsymposium on RNA Biology, 2022

Další údaje

Jazyk

angličtina

Typ výsledku

Konferenční abstrakt

Stát vydavatele

Česká republika

Utajení

není předmětem státního či obchodního tajemství

Organizační jednotka

Středoevropský technologický institut

Změněno: 21. 9. 2022 16:02, Mgr. Václav Hejret

Anotace

V originále

Mammalian cells express four AGO proteins (AGO1-4) that associate with miRNAs and act as effectors in RNAi pathways. Whereas miRNAs have long been considered the primary small RNA drivers of RNAi, several recent reports indicated that AGO proteins associate with small RNAs derived from other types of RNAs and suggested that they could serve similar functions like miRNAs. For instance, the CLASH (crosslinking, ligation, and sequencing of hybrids) analysis of AGO1 in Flp-In T-REx 293 cells uncovered a number of tRNA fragments (tRFs) and are predicted to confer post-transcriptional silencing regulation to their targets similar to miRNAs. Here we present the CLASH analysis of AGO2 in HEK293 cells to address the small RNA repertoire and uncover their physiological targets. We developed an optimized bioinformatics approach of chimeric read identification to detect chimeras of high confidence. In this new pipeline, we start with raw sequenced CLASH derived reads and the alignment to both whole genome and to specific noncoding RNA databases gives us potential to observe and annotate a wide scale of various AGO2 driven interactions. We identified 16828 chimeric target sites also supported by non-chimeric reads. 43% of these were miRNA:mRNA pairs. Interestingly, the majority of identified chimeric target interactions appears to be driven by non-miRNA driver molecule fragments (tRNA, rRNA, yRNA, snoRNA, vaultRNA). By using a combination of reporter assays and small RNA inhibitory assays we were able to show that at least some of these non-miRNA driver interactions regulate the steady state level of their targets determined by the CLASH. Identified small RNA:target chimeras were used to train Convolutional Neural Network models that can predict the potential of small RNA:target site binding with above state-of-the-art accuracy.

Návaznosti

GJ19-10976Y, projekt VaV

Název: Klasifikace miRNA vazebných míst nezávisle na „seed” oblasti

Investor: Grantová agentura ČR, Klasifikace miRNA vazebných míst nezávisle na „seed” oblasti

MUNI/A/1467/2021, interní kód MU

Název: Struktura a dynamika biopolymerů

Investor: Masarykova univerzita, Struktura a dynamika biopolymerů

Citovat

HEJRET, Václav, Nandan Mysore VARADARAJAN, Eva KLIMENTOVÁ, Ilektra-Chara GIASSA, Štěpánka VAŇÁČOVÁ a Panagiotis ALEXIOU. Noncanonical small RNA chimeras identified by AGO2-CLASH. In 16th Microsymposium on RNA Biology. 2022.

@proceedings{1855257,
   author = {Hejret, Václav and Varadarajan, Nandan Mysore and Klimentová, Eva and Giassa, IlektraandChara and Vaňáčová, Štěpánka and Alexiou, Panagiotis},
   booktitle = {16th Microsymposium on RNA Biology},
   language = {eng},
   title = {Noncanonical small RNA chimeras identified by AGO2-CLASH},
   year = {2022}
}

TY  - CONF
ID  - 1855257
AU  - Hejret, Václav - Varadarajan, Nandan Mysore - Klimentová, Eva - Giassa, Ilektra-Chara - Vaňáčová, Štěpánka - Alexiou, Panagiotis
PY  - 2022
TI  - Noncanonical small RNA chimeras identified by AGO2-CLASH
N2  - Mammalian cells express four AGO proteins (AGO1-4) that associate with miRNAs and act as effectors in RNAi pathways. Whereas miRNAs have long been considered the primary small RNA drivers of RNAi, several recent reports indicated that AGO proteins associate with small RNAs derived from other types of RNAs and suggested that they could serve similar functions like miRNAs. For instance, the CLASH (crosslinking, ligation, and sequencing of hybrids) analysis of AGO1 in Flp-In T-REx 293 cells uncovered a number of tRNA fragments (tRFs) and are predicted to confer post-transcriptional silencing regulation to their targets similar to miRNAs. Here we present the CLASH analysis of AGO2 in HEK293 cells to address the small RNA repertoire and uncover their physiological targets. We developed an optimized bioinformatics approach of chimeric read identification to detect chimeras of high confidence. In this new pipeline, we start with raw sequenced CLASH derived reads and the alignment to both whole genome and to specific noncoding RNA databases gives us potential to observe and annotate a wide scale of various AGO2 driven interactions. We identified 16828 chimeric target sites also supported by non-chimeric reads. 43% of these were miRNA:mRNA pairs. Interestingly, the majority of identified chimeric target interactions appears to be driven by non-miRNA driver molecule fragments (tRNA, rRNA, yRNA, snoRNA, vaultRNA). By using a combination of reporter assays and small RNA inhibitory assays we were able to show that at least some of these non-miRNA driver interactions regulate the steady state level of their targets determined by the CLASH. Identified small RNA:target chimeras were used to train Convolutional Neural Network models that can predict the potential of small RNA:target site binding with above state-of-the-art accuracy.
ER  -

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