Data-Independent Acquisition for the Detection of Mononucleoside RNA Modifications by Mass Spectrometry

RNA is dynamically modified in cells by a plethora of chemical moieties to modulate molecular functions and processes. Over 140 modifications have been identified across species and RNA types, with the highest density and diversity of modifications found in tRNA (tRNA). The methods used to identify and quantify these modifications have developed over recent years and continue to advance, primarily in the fields of next-generation sequencing (NGS) and mass spectrometry (MS). Most current NGS methods are limited to antibody-recognized or chemically derivatized modifications and have limitations in identifying multiple modifications simultaneously. Mass spectrometry can overcome both of these issues, accurately identifying a large number of modifications in a single run. Here, we present advances in MS data acquisition for the purpose of RNA modification identification and quantitation. Using this approach, we identified multiple tRNA wobble position modifications in Arabidopsis thaliana that are upregulated in salt-stressed growth conditions and may stabilize translation of salt stress induced proteins. This work presents improvements in methods for studying RNA modifications and introduces a possible regulatory role of wobble position modifications in A. thaliana translation

Data-Independent Acquisition for the Detection of Mononucleoside RNA Modifications by Mass Spectrometry

RNA is dynamically modified in cells by a plethora of chemical moieties to modulate molecular functions and processes. Over 140 modifications have been identified across species and RNA types, with the highest density and diversity of modifications found in tRNA (tRNA). The methods used to identify and quantify these modifications have developed over recent years and continue to advance, primarily in the fields of next-generation sequencing (NGS) and mass spectrometry (MS). Most current NGS methods are limited to antibody-recognized or chemically derivatized modifications and have limitations in identifying multiple modifications simultaneously. Mass spectrometry can overcome both of these issues, accurately identifying a large number of modifications in a single run. Here, we present advances in MS data acquisition for the purpose of RNA modification identification and quantitation. Using this approach, we identified multiple tRNA wobble position modifications in Arabidopsis thaliana that are upregulated in salt-stressed growth conditions and may stabilize translation of salt stress induced proteins. This work presents improvements in methods for studying RNA modifications and introduces a possible regulatory role of wobble position modifications in A. thaliana translation

Table_2_A Conserved Long Intergenic Non-coding RNA Containing snoRNA Sequences, lncCOBRA1, Affects Arabidopsis Germination and Development.XLSX

Long non-coding RNAs (lncRNAs) are an increasingly studied group of non-protein coding transcripts with a wide variety of molecular functions gaining attention for their roles in numerous biological processes. Nearly 6,000 lncRNAs have been identified in Arabidopsis thaliana but many have yet to be studied. Here, we examine a class of previously uncharacterized lncRNAs termed CONSERVED IN BRASSICA RAPA (lncCOBRA) transcripts that were previously identified for their high level of sequence conservation in the related crop species Brassica rapa, their nuclear-localization and protein-bound nature. In particular, we focus on lncCOBRA1 and demonstrate that its abundance is highly tissue and developmental specific, with particularly high levels early in germination. lncCOBRA1 contains two snoRNAs domains within it, making it the first sno-lincRNA example in a non-mammalian system. However, we find that it is processed differently than its mammalian counterparts. We further show that plants lacking lncCOBRA1 display patterns of delayed germination and are overall smaller than wild-type plants. Lastly, we identify the proteins that interact with lncCOBRA1 and propose a novel mechanism of lincRNA action in which it may act as a scaffold with the RACK1A protein to regulate germination and development, possibly through a role in ribosome biogenesis.</p

Image_6_A Conserved Long Intergenic Non-coding RNA Containing snoRNA Sequences, lncCOBRA1, Affects Arabidopsis Germination and Development.JPEG

Long non-coding RNAs (lncRNAs) are an increasingly studied group of non-protein coding transcripts with a wide variety of molecular functions gaining attention for their roles in numerous biological processes. Nearly 6,000 lncRNAs have been identified in Arabidopsis thaliana but many have yet to be studied. Here, we examine a class of previously uncharacterized lncRNAs termed CONSERVED IN BRASSICA RAPA (lncCOBRA) transcripts that were previously identified for their high level of sequence conservation in the related crop species Brassica rapa, their nuclear-localization and protein-bound nature. In particular, we focus on lncCOBRA1 and demonstrate that its abundance is highly tissue and developmental specific, with particularly high levels early in germination. lncCOBRA1 contains two snoRNAs domains within it, making it the first sno-lincRNA example in a non-mammalian system. However, we find that it is processed differently than its mammalian counterparts. We further show that plants lacking lncCOBRA1 display patterns of delayed germination and are overall smaller than wild-type plants. Lastly, we identify the proteins that interact with lncCOBRA1 and propose a novel mechanism of lincRNA action in which it may act as a scaffold with the RACK1A protein to regulate germination and development, possibly through a role in ribosome biogenesis.</p

Table_1_A Conserved Long Intergenic Non-coding RNA Containing snoRNA Sequences, lncCOBRA1, Affects Arabidopsis Germination and Development.XLSX

Long non-coding RNAs (lncRNAs) are an increasingly studied group of non-protein coding transcripts with a wide variety of molecular functions gaining attention for their roles in numerous biological processes. Nearly 6,000 lncRNAs have been identified in Arabidopsis thaliana but many have yet to be studied. Here, we examine a class of previously uncharacterized lncRNAs termed CONSERVED IN BRASSICA RAPA (lncCOBRA) transcripts that were previously identified for their high level of sequence conservation in the related crop species Brassica rapa, their nuclear-localization and protein-bound nature. In particular, we focus on lncCOBRA1 and demonstrate that its abundance is highly tissue and developmental specific, with particularly high levels early in germination. lncCOBRA1 contains two snoRNAs domains within it, making it the first sno-lincRNA example in a non-mammalian system. However, we find that it is processed differently than its mammalian counterparts. We further show that plants lacking lncCOBRA1 display patterns of delayed germination and are overall smaller than wild-type plants. Lastly, we identify the proteins that interact with lncCOBRA1 and propose a novel mechanism of lincRNA action in which it may act as a scaffold with the RACK1A protein to regulate germination and development, possibly through a role in ribosome biogenesis.</p

Image_2_A Conserved Long Intergenic Non-coding RNA Containing snoRNA Sequences, lncCOBRA1, Affects Arabidopsis Germination and Development.JPEG

Long non-coding RNAs (lncRNAs) are an increasingly studied group of non-protein coding transcripts with a wide variety of molecular functions gaining attention for their roles in numerous biological processes. Nearly 6,000 lncRNAs have been identified in Arabidopsis thaliana but many have yet to be studied. Here, we examine a class of previously uncharacterized lncRNAs termed CONSERVED IN BRASSICA RAPA (lncCOBRA) transcripts that were previously identified for their high level of sequence conservation in the related crop species Brassica rapa, their nuclear-localization and protein-bound nature. In particular, we focus on lncCOBRA1 and demonstrate that its abundance is highly tissue and developmental specific, with particularly high levels early in germination. lncCOBRA1 contains two snoRNAs domains within it, making it the first sno-lincRNA example in a non-mammalian system. However, we find that it is processed differently than its mammalian counterparts. We further show that plants lacking lncCOBRA1 display patterns of delayed germination and are overall smaller than wild-type plants. Lastly, we identify the proteins that interact with lncCOBRA1 and propose a novel mechanism of lincRNA action in which it may act as a scaffold with the RACK1A protein to regulate germination and development, possibly through a role in ribosome biogenesis.</p

Image_4_A Conserved Long Intergenic Non-coding RNA Containing snoRNA Sequences, lncCOBRA1, Affects Arabidopsis Germination and Development.JPEG

Long non-coding RNAs (lncRNAs) are an increasingly studied group of non-protein coding transcripts with a wide variety of molecular functions gaining attention for their roles in numerous biological processes. Nearly 6,000 lncRNAs have been identified in Arabidopsis thaliana but many have yet to be studied. Here, we examine a class of previously uncharacterized lncRNAs termed CONSERVED IN BRASSICA RAPA (lncCOBRA) transcripts that were previously identified for their high level of sequence conservation in the related crop species Brassica rapa, their nuclear-localization and protein-bound nature. In particular, we focus on lncCOBRA1 and demonstrate that its abundance is highly tissue and developmental specific, with particularly high levels early in germination. lncCOBRA1 contains two snoRNAs domains within it, making it the first sno-lincRNA example in a non-mammalian system. However, we find that it is processed differently than its mammalian counterparts. We further show that plants lacking lncCOBRA1 display patterns of delayed germination and are overall smaller than wild-type plants. Lastly, we identify the proteins that interact with lncCOBRA1 and propose a novel mechanism of lincRNA action in which it may act as a scaffold with the RACK1A protein to regulate germination and development, possibly through a role in ribosome biogenesis.</p

Image_1_A Conserved Long Intergenic Non-coding RNA Containing snoRNA Sequences, lncCOBRA1, Affects Arabidopsis Germination and Development.JPEG

Long non-coding RNAs (lncRNAs) are an increasingly studied group of non-protein coding transcripts with a wide variety of molecular functions gaining attention for their roles in numerous biological processes. Nearly 6,000 lncRNAs have been identified in Arabidopsis thaliana but many have yet to be studied. Here, we examine a class of previously uncharacterized lncRNAs termed CONSERVED IN BRASSICA RAPA (lncCOBRA) transcripts that were previously identified for their high level of sequence conservation in the related crop species Brassica rapa, their nuclear-localization and protein-bound nature. In particular, we focus on lncCOBRA1 and demonstrate that its abundance is highly tissue and developmental specific, with particularly high levels early in germination. lncCOBRA1 contains two snoRNAs domains within it, making it the first sno-lincRNA example in a non-mammalian system. However, we find that it is processed differently than its mammalian counterparts. We further show that plants lacking lncCOBRA1 display patterns of delayed germination and are overall smaller than wild-type plants. Lastly, we identify the proteins that interact with lncCOBRA1 and propose a novel mechanism of lincRNA action in which it may act as a scaffold with the RACK1A protein to regulate germination and development, possibly through a role in ribosome biogenesis.</p

Image_3_A Conserved Long Intergenic Non-coding RNA Containing snoRNA Sequences, lncCOBRA1, Affects Arabidopsis Germination and Development.JPEG

Long non-coding RNAs (lncRNAs) are an increasingly studied group of non-protein coding transcripts with a wide variety of molecular functions gaining attention for their roles in numerous biological processes. Nearly 6,000 lncRNAs have been identified in Arabidopsis thaliana but many have yet to be studied. Here, we examine a class of previously uncharacterized lncRNAs termed CONSERVED IN BRASSICA RAPA (lncCOBRA) transcripts that were previously identified for their high level of sequence conservation in the related crop species Brassica rapa, their nuclear-localization and protein-bound nature. In particular, we focus on lncCOBRA1 and demonstrate that its abundance is highly tissue and developmental specific, with particularly high levels early in germination. lncCOBRA1 contains two snoRNAs domains within it, making it the first sno-lincRNA example in a non-mammalian system. However, we find that it is processed differently than its mammalian counterparts. We further show that plants lacking lncCOBRA1 display patterns of delayed germination and are overall smaller than wild-type plants. Lastly, we identify the proteins that interact with lncCOBRA1 and propose a novel mechanism of lincRNA action in which it may act as a scaffold with the RACK1A protein to regulate germination and development, possibly through a role in ribosome biogenesis.</p

Table_3_A Conserved Long Intergenic Non-coding RNA Containing snoRNA Sequences, lncCOBRA1, Affects Arabidopsis Germination and Development.XLSX

Long non-coding RNAs (lncRNAs) are an increasingly studied group of non-protein coding transcripts with a wide variety of molecular functions gaining attention for their roles in numerous biological processes. Nearly 6,000 lncRNAs have been identified in Arabidopsis thaliana but many have yet to be studied. Here, we examine a class of previously uncharacterized lncRNAs termed CONSERVED IN BRASSICA RAPA (lncCOBRA) transcripts that were previously identified for their high level of sequence conservation in the related crop species Brassica rapa, their nuclear-localization and protein-bound nature. In particular, we focus on lncCOBRA1 and demonstrate that its abundance is highly tissue and developmental specific, with particularly high levels early in germination. lncCOBRA1 contains two snoRNAs domains within it, making it the first sno-lincRNA example in a non-mammalian system. However, we find that it is processed differently than its mammalian counterparts. We further show that plants lacking lncCOBRA1 display patterns of delayed germination and are overall smaller than wild-type plants. Lastly, we identify the proteins that interact with lncCOBRA1 and propose a novel mechanism of lincRNA action in which it may act as a scaffold with the RACK1A protein to regulate germination and development, possibly through a role in ribosome biogenesis.</p