Liu Huiquan's research group from Northwest A&F University created a comprehensive and complete full-length transcript annotation of Fusarium graminearum, revealing the regulatory functions of alternative splicing and variable polyadenylation

Editor-in-Chief | Wang Yi Recently, Liu Huiquan's research group from Northwest A&F University's School of Plant Protection/State Key Laboratory of Crop Stress Biology in Arid Regions published a research titled Landscape and regulation of alternative splicing and alternative polyadenylation in a plant pathogenic fungus in New Phytologist thesis
.

This paper uses a variety of technologies such as full-length transcriptome Iso-Seq, strand-specific RNA-Seq and gene function verification to create a comprehensive and complete set of filamentous plant pathogenic fungi, Fusarium graminearum.
Full-length transcripts were annotated, and the characteristics and regulatory functions of alternative splicing and alternative polyadenylation were systematically studied
.

The results of this study not only facilitate the in-depth study of F.
graminearum functional genomics, but also provide new insights into the complexity and regulation of alternative splicing and variable polyadenylation in filamentous fungi
.

The main findings and innovations achieved in this study are as follows: 1.
The assembly and sequence errors in the reference genome of F.
graminearum were corrected, and a comprehensive set of full-length transcript annotations was established for F.
graminearum
.

Fusarium graminearum is the main pathogen causing wheat scab.
Due to its scientific and economic importance and easy genetic manipulation, Fusarium graminearum has gradually become the model of functional genomics research of plant pathogenic fungi.
Up to now, Researchers at home and abroad have carried out knockout or mutation studies on at least 2,000 genes in the F.
graminearum genome
.

Comparative and functional genomics studies rely on precise genome sequences and gene/transcript annotations.
There are still many errors and imperfections in the existing F.
graminearum reference genome and gene annotation information, especially the existing gene annotations are mainly obtained through Computational predictions lack true transcript information, and most predicted genes lack noncoding regions (UTRs)
.

In view of this, this study corrected 249 errors in the existing reference genome of F.
graminearum PH-1 strain through third-generation PacBio genome sequencing assembly and second-generation Illumina resequencing, of which 7 were assembly errors and the rest were base mutations or indel errors
.

By performing third-generation PacBio full-length transcriptome (Iso-Seq) and second-generation strand-specific RNA-Seq sequencing analysis of samples from different tissues or stages of F.
Full-length transcript annotation of 51,617 transcript isoforms (Isoforms)
.

5481 long non-coding RNAs (lncRNAs) were also included in the full-length transcript annotation
.

Compared with the existing gene annotations (14145 genes, each gene has only one predicted transcript), the newly generated F.
graminearum gene/transcript annotation is greatly improved in terms of completeness and comprehensiveness
.

This is also the most comprehensive and complete transcript annotation reported in filamentous fungi
.

In order to facilitate domestic and foreign counterparts to use the new genome sequence and gene/transcript annotation of F.
graminearum, the research group has also built a F.
graminearum genome database website (http://fgbase.
wheatscab.
com/)
.

2.
To clarify the dynamics of alternative splicing and variable polyadenylation in F.
graminearum, and find that alternative splicing and variable polyadenylation regulate fungal tissue senescence and dormancy
.

Variable polyadenylation has been rarely studied in filamentous fungi, and alternative splicing has been studied in filamentous fungi.
However, RNA-Seq currently used for fungal alternative splicing studies is mostly non-strand-specific.

.

Due to the large gene density of fungi, overlapping of adjacent gene transcripts is common, and antisense RNA is unusually prevalent in some periods
.

Common non-strand-specific RNA-Seq cannot overcome the above problems, and it is more difficult to obtain accurate alternatively spliced ​​transcript isoforms
.

Identification of differential alternative splicing events in different samples often relies on complete alternative splicing transcript annotations
.

Using full-length transcript data, the study found that 43% of the intronic genes in F.
graminearum had alternative splicing events, with an average of 3 alternatively spliced ​​transcript isoforms per gene; 65% of the genes had alternative splicing events.
Variable polyadenylation, with an average of 9 variable polyadenylation sites per gene (producing transcripts of varying 3'-UTR lengths), suggesting that alternative splicing and variable polyadenylation are Occurrence is also as common as in plants and animals
.

The study identified differential alternative splicing and variable polyadenylation events in different stages of F.
graminearum infection, toxin production, sexual reproduction, asexual reproduction, and vegetative growth, and found alternative splicing and variable polyadenylation events.
The onset of acidification is distinctly tissue- and period-specific
.

Interestingly, we found a general increase in the abundance of transcripts with intron retention events and distal variable polyadenylation sites in senescent and dormant tissues, and these increases in transcripts are associated with RNA splicing factors.
and down-regulated expression of 3'-UTR processing factors
.

Functions related to autophagy, RNA metabolism, ubiquitination, etc.
are significantly enriched in these transcripts, and these functions have been reported to be related to aging in animals
.

Long 5'- or 3'-UTR transcripts are known to be less efficient in translation, either by intron retention or by the use of distal variable polyadenylation sites, which can render the 5'- or 3' of the transcripts - UTR growth
.

Therefore, alternative splicing and variable polyadenylation in F.
graminearum are likely to act as a unified cellular reprogramming mechanism responsible for reducing protein production in senescent and dormant tissues, thereby regulating tissue senescence and dormancy
.

3.
It was found that the mechanism of nonsense-mediated mRNA degradation (NMD) in Fusarium graminearum was not obviously coupled with alternative splicing, but instead performed translation regulation
.

NMD is a conserved RNA degradation mechanism in eukaryotes, mainly used to degrade transcripts containing premature stop codons, preventing the production of truncated proteins
.

In animals, plants and yeast, the NMD machinery is often coupled to alternative splicing for the degradation of prematurely terminated transcripts resulting from alternative splicing
.

This study found that more than 44% of alternatively spliced ​​transcript isoforms in F.
graminearum have shortened predicted ORFs at the 5' or 3' end due to premature termination introduced by alternative splicing events
.

Surprisingly, however, the abundance of these ORF-shortened transcripts did not increase in the NMD deletion mutant ΔFgupf1, indicating that these transcripts are not significantly regulated by NMD
.

Furthermore, analysis of the expression of alternatively spliced ​​transcripts with canonical NMD-inducible characteristics in ΔFgupf1 mutants also found no evidence of NMD regulation
.

In contrast, the differentially expressed transcripts in the ΔFgupf1 mutant were mainly canonical transcripts or alternatively spliced ​​transcripts with no altered ORF
.

Deletion of NMD in yeast (deletion of the UPF1 gene) did not cause significant growth defects, whereas the ΔFgupf1 mutant grew abnormally slowly
.

Interestingly, the down-regulated transcripts in ΔFgupf1 are mainly involved in ribosome biogenesis, suggesting that NMD may regulate gene expression mainly at the translational level in F.
graminearum
.

4.
It was found that the mRNA 3'-end processing factors FgRNA15, FgHRP1 and FgFIP1 in Fusarium graminearum not only regulate variable polyadenylation, but also regulate alternative splicing
.

The preference for which variable polyadenylation site to use is usually determined by the polyadenylation recognition signal (conserved recognition motif) and mRNA 3'-end processing factors
.

This study found that although the recognition signal of the distal variable polyadenylation site is stronger in F.
graminearum, it mainly uses the proximal variable polyadenylation site to express short 3'-UTR transcripts.
Different from reports in model yeast and animals
.

Through the functional study of the homologous encoding gene of the mRNA core 3'-end processing factor in F.
graminearum, it is proved that FgRNA15, FgHRP1, FgFIP1 promote the use of the proximal variable polyadenylation site in F.
graminearum, knocking The use of distal variable polyadenylation sites generally increased in all but mutants
.

Unexpectedly, intron retention events were significantly increased in these mutants, especially in the ΔFgRNA15 mutant, suggesting that these three 3'-terminal processing factors also regulate alternative splicing by promoting intron excision
.

The regulation of alternative splicing by 3'-terminal processing factors has only been reported in humans (Misra et al, Mol Cell, 2015, doi: 10.
1016/j.
molcel.
2015.
03.
016), and only CPF has been reported in humans : CPSF in the CFII subcomplex has this regulatory effect, other 3'-terminal processing factors do not
.

FgRNA15, FgHRP1, and FgFIP1 belong to CFIA, CFIB, and CPF:PFI subcomplexes.
Therefore, three different 3'-terminal processing factor subcomplexes in F.
graminearum all play a role in regulating alternative splicing
.

Lu Ping, a doctoral student at the School of Plant Protection, Northwest A&F University, is the first author of the paper, and Professor Liu Huiquan, a professor at the School of Plant Protection at Northwest A&F University and the State Key Laboratory of Crop Stress Biology in Arid Regions, is the corresponding author.
Professor Jiang Cong, Associate Professor Wang Qinhu and some graduate students from the scientific and technological innovation team also participated in the work
.

The research is funded by the National Key R&D Program and the National Natural Science Foundation of China
.

Paper link: https://nph.
onlinelibrary.
wiley.
com/doi/abs/10.
1111/nph.
18164