Transient expression of intron-containing transgenes generates non-spliced aberrant pre-mRNAs that are processed into siRNAs
Transient expression of intron-containing transgenes generates non-spliced aberrant pre-mRNAs that are processed into siRNAs
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DOI：
10.1007/s00425-018-3015-6
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发表时间：
2019-02-01
期刊：
影响因子：
4.3
通讯作者：
Wassenegger, Michael
中科院分区：
文献类型：
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作者：
Dalakouras, Athanasios;Lauter, Anja;Wassenegger, Michael
主要结论在本研究中，我们发现来自非剪接和非多聚腺苷酸化内含子转基因的异常前mRNAs被引导到RNA沉默途径。在植物中，处理不当的转录本被称为异常RNAs(ab-RNAs)，可以通过RNA沉默或RNA衰变机制来消除。从无内含子基因转录的AB-RNAs被RNA导向的RNA聚合酶(RDR)复制成双链RNA，然后再被Dice-like内切酶切割成小RNA(SRNAs)。相反，来自内含子基因的ab-RNAs被建议在剪接后引导到核外降解。然而，目前还不清楚非剪接异常的前mRNAs是如何被消除的。我们推测，农业渗透的含有内含子的转基因在本氏烟草中的瞬时表达将使我们能够研究非剪接前mRNAs的稳态水平。对农业浸润性转基因的sRNA深度测序显示，存在映射到整个非剪接前mRNA的sRNAs，这表明RDRs(很可能是RDR6)处理异常的非剪接前mRNA。检测到了长度为18-25个核苷酸(NT)的初级和次级sRNA，其中最显著的sRNA大小类为22nT。SRNA也映射到终止子序列，表明RDR底物也包含没有多聚腺苷酸化尾巴的直读转录本。重要的是，发生的sRNAs有效地靶向同源mRNA进行降解，但未能切割非剪接的Pre-mRNA，证实了sRNAs没有在细胞核中引发RNA切割的观点。
Main conclusionIn this study, we show that aberrant pre-mRNAs from non-spliced and non-polyadenylated intron-containing transgenes are channelled to the RNA silencing pathway.In plants, improperly processed transcripts are called aberrant RNAs (ab-RNAs) and are eliminated by either RNA silencing or RNA decay mechanisms. Ab-RNAs transcribed from intronless genes are copied by RNA-directed RNA polymerases (RDRs) into double-stranded RNAs which are subsequently cleaved by DICER-LIKE endonucleases into small RNAs (sRNAs). In contrast, ab-RNAs from intron-containing genes are suggested to be channelled post-splicing to exonucleolytic degradation. Yet, it is not clear how non-spliced aberrant pre-mRNAs are eliminated. We reasoned that transient expression of agroinfiltrated intron-containing transgenes in Nicotiana benthamiana would allow us to study the steady-state levels of non-spliced pre-mRNAs. SRNA deep sequencing of the agroinfiltrated transgenes revealed the presence of sRNAs mapping to the entire non-spliced pre-mRNA suggesting that RDRs (most likely RDR6) processed aberrant non-spliced pre-mRNAs. Primary and secondary sRNAs with lengths of 18-25 nucleotides (nt) were detected, with the most prominent sRNA size class of 22nt. SRNAs also mapped to the terminator sequence, indicating that RDR substrates also comprised read-through transcripts devoid of polyadenylation tail. Importantly, the occurring sRNAs efficiently targeted cognate mRNA for degradation but failed to cleave the non-spliced pre-mRNA, corroborating the notion that sRNAs are not triggering RNA cleavage in the nucleus.