基于高通量测序鉴定细菌RNA加工位点方法的开发（英文）-豆柴文库

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基于高通量测序鉴定细菌RNA加工位点方法的开发（英文）
DevelopmentofaHigh-ThroughputSequencing-BasedMethodforIdentificationofBacterialRNAProcessingSites
Introduction
Bacteriahaveevolvedamultitudeofmechanismstoregulategeneexpressionatthepost-transcriptionallevel.TheseincludeRNAprocessing,degradation,andqualitycontrol,whichcanhaveasignificantimpactonthestability,translation,andfunctionofmRNAmolecules.OneofthekeystepsinRNAprocessingisthecleavageandpolyadenylationofpre-mRNAtogeneratematuremRNA,whichcanbetranslatedintoprotein.Inmostbacteria,thisprocessiscatalyzedbytheRNAdegradosome,alargeproteincomplexthatcontainsmultipleRNAprocessingenzymes.However,thepreciselocationofRNAcleavageandpolyadenylationsitesremainslargelyunknowninmanybacterialspecies.
Methods
Toaddressthischallenge,wehavedevelopedahigh-throughputsequencing-basedmethodforidentifyingbacterialRNAprocessingsites.Thisapproachinvolvesthefollowingsteps:
1.RNAisolationandlibrarypreparation:TotalRNAisisolatedfrombacterialcellsusingstandardmethodsandsubjectedtopoly(A)selectiontoenrichformRNAmolecules.Thepoly(A)-tailedmRNAisthenfragmentedandreversetranscribedintocDNA,whichisfurtheramplifiedbyPCRtogenerateasequencinglibrary.
2.High-throughputsequencing:Thelibraryissubjectedtohigh-throughputsequencingusingIlluminaorotherplatforms,generatingmillionsofshortreadsthatspantheentiretranscriptome.
3.Dataanalysis:Thesequencingdataisthenanalyzedusingacustombioinformaticspipeline.Thereadsarealignedtothereferencegenome,andthe3’endpositionsofthereadsareusedtoinfertheRNAprocessingsites.Thedataisthenfilteredtoremovelow-qualityreads,andtheresultingRNAprocessingsitesarecomparedtoknowngeneannotationstoidentifynovelsites.
Results
Usingthismethod,wehavegeneratedhigh-qualitysequencingdatafromseveralbacterialspecies,includingEscherichiacoliandPseudomonasaeruginosa.WehaveidentifiednumerousnovelRNAprocessingsitesinthesespecies,includingboth3’and5’processingsites.Manyofthesesiteswerelocatedinthecodingregionofgenes,suggestingthatRNAprocessingplaysanimportantroleinregulatin