Mechanistic insights into human pre-mRNA splicing of human ultra-short introns: Potential unusual mechanism identifies G-rich introns

https://doi.org/10.1016/j.bbrc.2012年05月11日2 Get rights and content

Abstract

It is unknown how very short introns (<65 nt; termed ‘ultra-short’ introns) could be spliced in a massive spliceosome (>2.7 MDa) without steric hindrance. By screening an annotated human transcriptome database (H-InvDB), we identified three model ultra-short introns: the 56-nt intron in the HNRNPH1 (hnRNP H1) gene, the 49-nt intron in the NDOR1 (NADPH dependent diflavin oxidoreductase 1) gene, and the 43-nt intron in the ESRP2 (epithelial splicing regulatory protein 2) gene. We verified that these endogenous ultra-short introns are spliced, and also recapitulated this in cultured cells transfected with the corresponding mini-genes. The splicing of these ultra-short introns was repressed by a splicing inhibitor, spliceostatin A, suggesting that SF3b (a U2 snRNP component) is involved in their splicing processes. The 56-nt intron containing a pyrimidine-rich tract was spliced out in a lariat form, and this splicing was inhibited by the disruption of U1, U2, or U4 snRNA. In contrast, the 49- and 43-nt introns were purine-rich overall without any pyrimidine-rich tract, and these lariat RNAs were not detectable. Remarkably, shared G-rich intronic sequences in the 49- and 43-nt introns were required for their splicing, suggesting that these ultra-short introns may recruit a novel auxiliary splicing mechanism linked to G-rich intronic splicing enhancers.

Highlights

► We identified and characterized ultra-short introns (<65-nt) of human pre-mRNAs. ► The 56-, 49-, and 43-nt introns were found in the HNRNPH1, NDOR1 , and ESRP2 genes. ► SF3b (U2 snRNP component) is involved in splicing of these 3 ultra-short introns. ► Splicing of the 56-nt intron requires the U1, U2, and U4 snRNPs. ► Splicing of the 49- and 43-nt introns requires G-rich intronic splicing enhancers.

Introduction

The length distributions of vertebrate pre-mRNA introns have a bimodal pattern, e.g., a narrow distribution peaking around ∼100 nucleotides (nt) (‘short introns’) and a broad distribution peaking around ∼2000 nt (‘long introns’) in humans [1], [2]. Although intron-definition and exon-definition models have been proposed for the splicing of short and long introns, respectively [3], we do not know much about the exact splicing mechanisms for introns in extra-range below and above of the bimodal distribution. In fact, the mechanism of pre-mRNA spicing has been studied with model pre-mRNAs containing single short introns (100–250 nt), which have been spliced efficiently in vivo and in vitro (reviewed in [4], [5]).
Here, we focus on very short introns in the range below the narrow distribution of the first mode, i.e., less than 65 nt in length, which we designated ‘ultra-short introns’ (Shimada et al., manuscript in preparation). Early studies showed that a minimum intron length of 78–80 nt is necessary for their splicing [6], [7]. However, the real minimum length cannot be inferred from these data because these tested introns were arbitrarily shortened from natural introns, which would eliminate important cis-acting elements [8], [9]. We carefully screened human genes containing functional ultra-short introns and the splicing of identified three model ultra-short introns (43, 49, and 56 nt) was verified in vivo and in vitro.
Human nuclear pre-mRNA splicing involves dynamic stepwise reactions in a huge spliceosome, which includes five kinds of U snRNPs and ∼170 proteins (reviewed in [5], [10]). Essential cis-reactive elements in the pre-mRNA; the 5′ splice site, the branch-point sequence, and the 3′ splice site, are simultaneously bound by corresponding trans-acting factors, U1 snRNP, U2 snRNP, and U2AF65/U2AF35, respectively, leading to the initial ATP-dependent formation of the spliceosomal A complex (∼2.5 MDa) [11]. Structural analysis by electron microscopy has estimated that the A complex has an asymmetric globular shape, with dimensions of ∼26 ×ばつ 20 ×ばつ 19.5 nm [11]. Thus, the A complex fully occupies the length of an 85–113-nt linearized RNA (as 1-nt RNA = 0.23 nm; reference [12]), which is about twofold longer than the ultra-short introns we tested (43–56 nt). Therefore, we asked how these three essential splicing signals can be recognized by the corresponding essential factors without steric hindrance.

Section snippets

Materials and methods

Full descriptions are provided in the Supplementary Materials and methods.

Human ultra-short introns were screened and identified

Using the intronic sequences from the human transcript database (H-InvDB, version 6.0) and the human genome database (NCBI build 36), we derived the pattern of the length distribution of the human introns that are shorter than 945 nt (Fig. 1A, left panel). We observed that the number of human introns increases drastically from 65 nt toward the first mode of the distribution at 83 nt, which is essentially consistent with previous reports [1], [2]. Most of these short introns could be spliced by

Discussion

Here, we report the first identification and characterization of bona fide ultra-short human introns (56, 49, and 43 nt) in the HNRNPH1, NDOR1, and ESRP2 genes, respectively. The functional proteins NDOR1 (diflavin oxidoreductase) and ESRP2 (epithelial splicing regulatory protein) are produced by the use of the distal 3′ splice sites in their pre-mRNAs. Utilization of the proximal 3′ splice site of NDOR1 (excision of the 49-nt intron) generates a protein isoform (NDOR1_v1) with a 90% reduction

Acknowledgments

We are grateful to Drs. M. Yoshida and A. Malhotra for providing the spliceostatin A and the purified recombinant RNase R, respectively. We thank Drs. A.R. Krainer and R. Yoshimoto for their valuable suggestions, and members of our laboratory for their support and discussions. This work was funded by research grants from the Hori Information Science Promotion Foundation to N.S-H. and the Okawa Foundation for Information and Telecommunications to A.M.

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