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Dissecting the role of alternative splicing in the regulation of autophagy: a narrative review

Zhang, Mengjiao; Zhang, Yingmei

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doi: 10.1097/JBR.0000000000000062

Abstract

Introduction

Alternative splicing is considered an essential machinery responsible for transcriptional diversity. This mechanism usually gives rise to proteomic complexity even with a limited number of genes. Changes of mRNA splicing have been perceived to play a key role in the onset and development of various pathological settings including coronary heart diseases, atherosclerosis and hypertension.[1,2] For example, heart failure may be associated with altered alternative splicing.[2,3] The alternative splicing process is controlled by a large ribonucleoprotein complex namely spliceosome, which consists of >100 core proteins and 5 small nuclear RNAs (U1, U2, U4, U5, and U6). Each of the small nuclear RNAs uses surrounding proteins to generate a small nuclear ribonucleoprotein.[3,4] A small nuclear ribonucleoprotein is capable of identifying core splicing codons in every intron including the 5′ splice site (containing the GU nucleotides), the 3′ splice site (containing the AG nucleotides and the polypyrimidine tract) along with the branch point sequence. Pre-mRNA splicing is reported to be executed through the removal of introns and the connection of exons together using spliceosome.[5,6] Alterations in exon exclusion, intron retention or the site of splicing are all contributing factors to the ultimate structure, localization and function of the protein profile.[6] In addition to spliceosome activity, recognition and regulation of alternative splicing may also be affected by additional sequences commonly known as auxiliary cis-regulatory sequences. Many experimental findings have indicated the localization of these auxiliary cis-regulatory sequences in exons and neighboring introns. It is believed that the auxiliary sequences may call upon trans-regulatory factors including Ser/Arg-rich proteins and heterogeneous nuclear ribonucleoproteins to accomplish alternative splicing of the entire transcript cascade. Research has indicated that Ser/Arg-rich proteins play an essential role in initiating the assembly of spliceosome, possibly by way of phosphorylation.[7] Various mechanisms have been proposed for these trans-regulatory factors, including controlling exclusive splicing response either independently, in cooperation or antagonistically.[8]

Autophagy, a cellular process for degrading and recycling of intracellular components, is composed of three main subtypes including macroautophagy, chaperone-mediated autophagy and microautophagy.[2,6] Autophagy is carried out by the formation of an autophagosome, a double-membraned cellular structure. Autophagy is a non-selective process distinct from the highly specific ubiquitin-proteasome system, which identifies the ubiquitinated proteins for proteasomal degradation.[9] Autophagy possesses a wide spectrum of biological functions such as starvation adaptation, development, aging retardation, cell death, tumor suppression, elimination of microorganisms and intracellular protein and organelle clearance.[10] Under physiological conditions, autophagy helps to maintain cellular homeostasis through the degradation of damaged or superfluous organelles.[11] Although autophagy is deemed protective within a physiological range, excess autophagic degradation can be devastating. Either too much or too little autophagy has been linked with a variety of human diseases including heart disease, metabolic diseases and diabetes mellitus.[12] Autophagy consists of several sequential steps including sequestration, degradation, and amino acid generation. In yeast, 31 autophagy-related (Atg) proteins have been identified. Among the 31 Atg proteins, 18 Atg proteins, Atg 1–10, Atg 12–14, Atg 16–18, Atg 29 and Atg 31, are involved in autophagosome formation (Fig. 1A). This review highlights recent advances in the understanding of mechanisms by which alternative splicing affects the functions of ATG genes including BECN1, ATG5, ATG16L1 and Bim genes, and thus manipulates autophagy levels in some diseases. These are collated in Table 1.

Figure 1
Figure 1:
The association between alternative splicing of the ATG genes and autophagy. (A) The overall process of macroautophagy. (B) The outcome of the splicing profile of BECN gene. BECN1 s is unable to recognize the coiled–coil UVRAG, thus retains the ability to activate PtdIns3K, which is important for initiation of autophagy. (C) The outcome of the splicing profile of ATG5 gene. Alternative splicing of ATG5 mRNA results in nonfunctional truncated products, ATG12–ATG5 conjugate was undetected, thus the levels of autophagy decreased. (D) The outcome of the splicing profile of ATG16L gene. Despite forming the ∼800-kDa complex with Atg12–5 and having E3-like activity for LC3, Atg16L2 is not recruited to phagophores. Atg = autophagy-related, BECN1s = BECN1 short isoform, LC3 = microtubule-associated proteins light chain 3, PtdIns3K = class III phosphatidylinositol 3-kinase, UVRAG = ultraviolet irradiation resistance-associated protein.
Table 1
Table 1:
Summary of the genetic variants, the action of translated proteins and functional outcome.

Mitochondrial injury imposes unfavorable outcomes for cells and organisms and is associated with an array of human diseases including aging, cardiovascular and autoimmune diseases.[13] Selective autophagy of mitochondria, mitophagy, governs target clearance of damaged mitochondria and helps to maintain the quality of mitochondria. Mitophagy can be triggered under many conditions including starvation, hypoxia, reactive oxygen species production and mitochondrial uncoupler-induced mitochondrial depolarization. Several mitophagy components have been identified such as PTEN-induced putative kinase 1 (Pink1)-Parkin, FUN14 domain-containing protein 1 (FundC1) and Bcl-2/adenovirus E1B 19-kDa-interacting protein (BNIP3). These receptors orchestrate the efficient clearance of long-lived or damaged mitochondria. To date, little information is readily available on alternative splicing control in autophagy and mitophagy (Additional file: Database search strategy, http://links.lww.com/JR9/A19). It is speculated that most of the ATG genes undergo alternative splicing and maintain several splicing variants.

Alternative splicing of BECN1 functions in autophagy and mitophagy

Beclin 1 is the primary mammalian autophagy protein, encoded by BECN1 gene, and is found in human B-cell acute lymphoblastic leukemia cells. Beclin 1 encoded by BECN1 gene, is the primary mammalian autophagy protein. Beclin 1 plays a cardinal role in autophagy. Evidence from human studies suggested that low Beclin 1 levels were linked with poor overall survival and the progression-free survival of natural T-cell lymphoma and non-Hodgkin lymphoma.[13,14] These findings indicated a key role for Beclin1 in lymphoid cell development and differentiation. In a recent study examining alternative splicing of BECN1 in acute lymphoblastic leukemia cells, a novel transcript variant of BECN1 was identified by DNA sequencing.[15] This variant of the Beclin 1 gene, due to alternative 3′ splicing displays a deletion of exon 11, resulted in C-terminal truncation of Beclin 1 and the loss of autophagy response to starvation. These findings favor an inhibitory effect of Beclin1 alternative splicing in autophagy.

In addition to exon 11 deletion, another splice variant of BECN1 has been reported.[16] The newly isoform was designated as BECN1 s (for short isoform), losing exon 10 and 11 of unspliced BECN1, and was believed to play a role in outer-membrane function. On the one hand, BECN1/Beclin 1 is well known to initiate autophagy through interaction with class III phosphatidylinositol 3-kinase (PtdIns3K). The binding affinity between the BECN1 s and PtdIns3K is intact, although BECN1 s would lose its ability to initiate autophagy. This is largely because BECN1 s is unable to recognize the coiled–coil ultraviolet irradiation resistance-associated protein (UVRAG), an indispensable component of the BECN1-PtdIns3K lipid kinase complex, thus resulting in failure to initiate autophagy (Fig. 1B). Intriguingly, despite BECN1 s not being obligatory for autophagy initiation, it is indispensable for starvation- and mitochondrial depolarization-induced mitophagy. Further evidence revealed a tight interaction between BECN1 s and PINK1, but not with heat shock protein 90, FUNDC1 and BNIP3, suggesting a Pink1-Parkin dependent mechanism for BECN1s-induced mitophagy.

Alternative splicing of ATG5 and ATG16 function in autophagy

In a study of valproic acid-induced autophagy in human prostate cancer cells, a distinct splicing profile of ATG5 gene was noted where valproic acid induces microtubule-associated protein 1A/1B-light chain 3 (LC3) formation in LNCaP and PC-3 cancer cells.[17] However, these autophagy markers were undetectable in DU145 cancer cells upon valproic acid induced autophagic stimulation. The defect of autophagy in this cell line was later found was associated with the mis-splicing of ATG5 gene. In the DU145 cells, a couple of alternative spliced ATG5 transcripts were observed. It was concluded that loss of one or two exons in these transcripts results in early termination of ATG5 translation. The truncated form of ATG5 cannot be conjugated with ATG12, leading to the impairment of autophagy pathway in DU145 cells (Fig. 1C).

In the autophagy elongation phase, several mammalian ATG genes namely ATG5, ATG12 and ATG16L1, form a tight complex. To understand the precise function of the ATG12-5-16L1 complex, considerable attention has been directed toward ATG16L1. A second isoform of mammalian ATG16L, termed ATG16L2, has been noted,[18] which can bind to ATG5, in a manner similar to ATG16L1. Nonetheless, the complex of ATG12-5-16L2 was unable to be recruited to phagophores. This finding indicates that the formation of such complex is inadequate to foster autophagosome formation (Fig. 1D). The same report showed that ATG16L2 was unnecessary for starvation-induced autophagy and was present at only a tenth the amount of ATG16L1.

Alternative splicing of Bim functions in autophagy and apoptosis

The process of apoptotic cell death is mediated by the Bcl-2 family that comprises of pro-survival proteins, pro-apoptotic members, and Bcl-2 homologue region 3 (BH3)-only proteins.[19] Among the BH3-only proteins, Bim has been known as a key regulator for T-cell apoptosis. Loss of Bim resulted in delayed apoptosis, as well as the impeded degradation of autophagy.[20] Analysis of gene splicing revealed three main isoforms for Bim namely BimEL, BimL and BimS (Fig. 2A). BimL facilitates lysosomal positioning through interaction with dynein that promote autolysosome formation in late lysosomal degradation. Excess BimL in Bim deficient cells stimulated vesicular aggregation, while other isoforms, BimEL and BimS, promoted intrinsic cell death (Fig. 2B). Given the distinctive properties of these three isoforms, the accurate alternative splicing of Bim gene is crucial to the control of apoptosis and autophagy.

Figure 2
Figure 2:
Alternative splicing of Bim functions in autophagy and apoptosis. (A) Alignment of human Bim isoforms and their transcripts. (B) The mechanism of BimS, BimL and BimEL function. With certain apoptotic stimuli, BimS and BimEL are bound to the antiapoptotic proteins Bcl-2 and Bcl-x(L) to neutralize their antiapoptotic activities in T cells. In the presence of BimL isoforms, the interaction of BimL with dynein facilitates the loading and perhaps fusion and positioning of lysosomes, which has a non-apoptotic activity. Adapted from Gross et al[19] and Ruppert et al,[20] respectively.

Conclusion

The mis-splicing of ATG genes would be expected to lead to the disruption of autophagy within cells. Most of the identified splicing variants of ATG genes resulted in failure to facilitate autophagy. Since autophagy is indispensable for the degradation of damaged or superfluous organelles in cells, maintaining autophagy within a tightly controlled range would be essential for cellular function. However, research into the relationships between alternative splicing and autophagy or mitophagy is still in its infancy. Among the hundreds of potential autophagy-associated proteins which might undergo splicing, only a few have been studied so far.

The regulation of alternative splicing is usually governed by trans-regulator splicing factors. Each regulatory protein can affect various RNA targets, while each transcript may be targeted by multiple regulators. The complexity of these regulatory pathways enables a high degree of specificity for where and when splicing events may occur. To-date, no particular splicing factors have been identified in the regulation of alternative splicing of autophagy or mitophagy genes. Thus, a better understanding of the transcriptional and post-transcriptional regulation of splicing factors is pertinent to explore unknown aspects of pathological conditions resulting from defects in autophagy and mitophagy. It is also important to characterize the different roles of protein isoforms and the regulatory mechanisms through a multidisciplinary approach, including genetics, biochemistry and physiology. New techniques give additional research avenues, such as next-generation RNA sequencing (RNAseq) that has emerged as an efficient tool to identify the new transcripts in total RNA. Alternative splicing would be a novel and promising field to explore the precise role of autophagy and mitophagy.

Acknowledgments

None.

Author contributions

MZ searched the references and writed the first draft. YZ revised the manuscript.

Financial support

This work was supported by the National Natural Science Foundation of China, No. 81770261 and Medical and Health Science and Technology Innovation Project of Chinese Academy of Medical Sciences, No. 2019-RC-HL-021.

Conflicts of interest

The authors declare that they have no conflicts of interest.

References

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Keywords:

alternative splicing; ATG gene; autophagy; mitochondria; mitophagy

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