NEW YORK (GenomeWeb News) – New research is providing evidence for unanticipated links between pathways governing RNA metabolism and those involved in RNA silencing in Arabidopsis.
In a paper published online yesterday in the early edition of the journal Developmental Cell, researchers from the Salk Institute and the University of Western Australia used a genome-wide approach to show that two RNA metabolism proteins also contribute to RNA silencing pathways affecting Arabidopsis development. Perhaps even more surprisingly, they found that one of these proteins influences a class of small RNAs produced from functional, annotated transcripts.
The team made the connections while investigating mutant plants lacking the RNA metabolism proteins ABH1, EIN5, or both. ABH1 (also known as CBP80), is the large sub-unit of the mRNA cap binding complex. EIN5 (also called XRN4), on the other hand, is a 5’ to 3’ exoribonuclease that degrades specific mRNA transcripts.
Neither protein had been linked to RNA silencing in the past. But the researchers noticed that the ein5 and/or abh1 mutants had phenotypes similar to those of plants with defects in their RNA-silencing pathway.
“It kind of pushed us down the RNA-silencing road,” lead author Brian Gregory, a post-doctoral researcher in plant molecular and cell biologist Joseph Ecker’s lab at the Salk Institute, told GenomeWeb Daily News.
In an attempt to understand whether the proteins influenced Arabidopsis’ smRNAome — smRNAs across the genome — the researchers analyzed the Arabidopsis transcriptome of wild type plants or mutants lacking ABH1, EIN5, or both, using whole-genome tiling microarrays. They then validated their results using reverse transcription quantitative PCR.
The team also did deep sequencing of the smRNAs found in the immature flower buds of the wild type and mutant plants using an Illumina Genome Analyzer.
Although ABH1 is best known for its roles in processes such as RNA maturation and surveillance, Gregory explained, the team discovered that ABH1 also affects miRNA levels in Arabidopsis cells. When ABH1 was absent, the level of mature miRNAs decreased, indicating that the protein contributes to miRNA-mediated RNA silencing.
“This is the first time that we’ve seen that [ABH1] has effects on the small RNA population,” Gregory said.
As for EIN5, Gregory and his colleagues found that the protein affects the level of a different group of smRNAs — including those formed from functional RNAs. For example, when EIN5 was absent, smRNAs tended to accumulate in parts of the genome containing euchromatin. When they looked more closely at 133 functionally annotated gene transcripts, the researchers found that they seemed to be processed into smRNAs.
“[C]lusters of mostly 21 nucleotide smRNAs were generated from both sense and antisense strands of these 133 functionally annotated transcripts, suggesting that these mRNAs have been converted to dsRNA and then processed in 21 nucleotide increments in the absence of functional EIN5,” the authors wrote.
“This was probably the most surprising finding of the work,” Gregory said. While it remains to be seen whether these smRNAs are functional, he added, the “EIN5-affected small RNAs” seem to be processed “like crazy” when EIN5 is absent.
Using a sequencing method that they developed to detect uncapped 5’ RNA ends, the researchers found that roughly half of the EIN5-affected smRNA-generating transcripts accumulated as uncapped transcripts when EIN5 was absent.
“[O]ur results suggest that an additional fate for endogenous uncapped transcripts is shuttling into an RNA silencing pathway where they become smRNA-biogenesis substrates,” the authors wrote. “Overall, these findings suggest that there is still much to learn concerning RNA silencing and the regulation of these post-transcriptional regulatory pathways.”
In the future, the team plans to use computational methods to look for potential targets for the new smRNAs, which they will then try to validate experimentally, Gregory said. In addition, he said, since ABH1 and EIN5 are conserved from yeast to humans, the study may ultimately provide insights into RNA metabolism and silencing in other organisms as well.