Discovery of a subset of human short introns spliced out by a distinct mechanism

Protein-coding genes carry the blueprint for protein production. In higher organisms, however, most of the coding-gene transcripts, or pre-mRNAs, are separated by non-coding sequences called “introns,” which must be  cut out or “spliced” to make mature mRNA that can be translated into protein.

Human pre-mRNA introns vary extensively in their lengths, ranging from under fifty to over a million nucleotides (nt). Human pre-mRNA splicing involves dynamic stepwise reactions in a huge protein-RNA complex called “spliceosome,” which includes five kinds of small nuclear ribonucleoproteins, called U snRNPs, and many protein factors. The essential splicing signal sequences in pre-mRNA—the 5’ splice site, the branch-site sequence, and the poly-pyrimidine tract (PPT) followed by the 3′ splice site—are bound by the splicing factors U1 snRNP, U2 snRNP, and U2AF⁶⁵/U2AF³⁵, respectively, which together constitute the spliceosomal A complex. The globular shape of the A complex fully occupies the length of a 79–125 nt single-stranded RNA, which is about two-fold longer than the known short introns (43–56 nt). How are these short introns able to accommodate the oversized complex with the known essential factors? It may be assumed that such short introns are spliced out by alternate mechanisms.

Now, a team of researchers led by Professor Akila Mayeda from the Institute for Comprehensive Medical Science, Fujita Health University, Japan, has attempted to answer this question in their latest study published in Nature Communications. Elaborating their findings, the paper’s co-author Kazuhiro Fukumura says, “The length variation of human pre-mRNA introns is extensive, ranging from fifty to over a million nucleotides. We thus postulate that there is possibly a distinct alternate splicing mechanism involved in splicing of human short introns.”

The team began by searching for essential factors to splice out human short introns from 154 human nuclear proteins. They downregulated these proteins’ expression in a human cell line (HeLa cells) using small interfering RNAs (siRNA). To analyze splicing activity, they selected HNRNPH1 pre-mRNA (heterogeneous nuclear ribonucleoprotein H1) including a 56-nt short intron.

The strongest splicing repression in HNRNPH1 pre-mRNA with 56-nt intron was caused by knockdown of SPF45, but no splicing repression was observed in pre-mRNA with control 366-nt intron. To further confirm that SPF45 is a common splicing factor for a group of short introns, they performed whole-transcriptome sequencing with RNA prepared from the SPF45-knockdown cells. The most frequent changes of splicing in SPF45-knockdown cells were intron retention, and 187 of the retained introns were identified. Remarkably, the length distribution of these SPF45-dependent introns was strongly biased towards shorter lengths. This suggested that SPF45 is required for the splicing of many pre-mRNAs with short introns.

Next, the researchers investigated the factor that determined the SPF45-dependence of some short introns. A PPT sequence and the downstream 3′ splice site is required for binding of the known authentic splicing factor U2AF heterodimer (U2AF⁶⁵/U2AF³⁵). Notably, a truncation in this PPT led to SPF45-dependency, suggesting that short PPT is crucial for SPF45-dependent splicing. As expected, a knockdown of the U2AF heterodimer significantly decreased the splicing of conventional introns; SPF45-dependent short introns, however, were spliced out rather efficiently, suggesting that SPF45 expels U2AF heterodimer on truncated PPTs and the newly installed SPF45 promotes short intron splicing. Finally, biochemical analyses and splicing assays with various mutant SPF45 proteins helped establish the model of SPF45-dependent splicing on a short intron with a truncated PPT (Figure 1).

Previously, SPF45 was reported to function as a regulator of alternative splicing; however, SPF45 is also an essential factor for cell survival and maintenance in vivo. The research team offers a solution to this enigma by demonstrating that SPF45 is a novel and distinct constitutive splicing factor in the early spliceosome, i.e., a subset of human short introns with truncated PPTs is spliced out with SPF45 but not with previously known authentic U2AF heterodimer.

Prof. Mayeda states, “This is a ground-breaking accomplishment in terms of basic research; however, the applications of our findings are also potentially intriguing. Overexpression of SPF45 confers multidrug resistance to anticancer drugs. Presumably, the genes involved in this mechanism harbor SPF45-dependent introns. Thus, overexpression of SPF45 may cause up-regulation of such genes though splicing activation of the transcripts. Understanding these mechanisms can aid in development of effective therapeutic interventions.”

***

Reference

Title of original paper: SPF45/RBM17-dependent, but not U2AF dependent, splicing in a distinct subset of human short introns

Journal: Nature Communications

DOI: https://doi.org/10.1038/s41467-021-24879-y

About Fujita Health University

Fujita Health University is a private university situated in Toyoake, Aichi, Japan. It was founded in 1964 and houses one of the largest teaching university hospitals in Japan in terms of the number of beds. With over 900 faculty members, the university is committed to providing various academic opportunities to students internationally. Fujita Health University has been ranked eighth among all universities and second among all private universities in Japan in the 2020 Times Higher Education (THE) World University Rankings. THE University Impact Rankings 2019 visualized university initiatives for sustainable development goals (SDGs). For the “good health and well-being” SDG, Fujita Health University was ranked second among all universities and number one among private universities in Japan. The university was the first Japanese university to host the "THE Asia Universities Summit" in June 2021, and also appointed as the host university in 2022. The university’s founding philosophy is “Our creativity for the people (DOKUSOU-ICHIRI),” which reflects the belief that, as with the university’s alumni and alumnae, current students also unlock their future by leveraging their creativity.

Website: https://www.fujita-hu.ac.jp/en/index.html

About Professor Akila Mayeda from Fujita Health University

Dr. Akila Mayeda ran his own lab at the University of Miami School of Medicine from 1998 to 2007. Since 2007, he has been a tenured Professor at the Division of Gene Expression Mechanism, Institute for Comprehensive Medical Science, Fujita Health University. He is one of the pioneers of human pre-mRNA splicing studies, and internationally recognized leader in the field of the mechanism of human gene expression. To date, he has authored 82 publications across these areas in international journals. His research team is currently investigating the mRNA quality control mechanisms in pre-mRNA splicing and the mechanism of aberrant splicing and splicing defects involved in cancer pathologies.

Website: http://www.fujita-hu.ac.jp/~mayeda/index-e.html

Funding information

Grants-in-Aid for Scientific Research (C)/(B) [Grant numbers: 18K07304, JP16H04705] and for Challenging Exploratory Research [Grant number: JP16K14659] from the Japan Society for the Promotion of Science (JSPS).