The 5′ UTR intron-mediated enhancement of constitutive splicing of the tobacco microsome ω-3 fatty acid desaturase gene

Sayuri Ohta,Sakie Nakagawara,Sayaka Hirai,Kumi Miyagishima,Gorou Horiguchi,Hiroaki Kodama 한국식물생명공학회 2018 Plant biotechnology reports Vol.12 No.2

Several plant genes have their first intron in the 5′ untranslated region (5′ UTR), and such 5′ UTR introns often show several biological functions, including the intron-mediated enhancement of protein expression through an increase of mRNA level (IME), intron-dependent spatial expression, and intron-mediated enhancement of translation. Here, we show another function of the 5′ UTR intron, i.e., the 5′ UTR intron-mediated enhancement of constitutive splicing. The NtFAD3 gene, which encodes a tobacco microsome ω-3 fatty acid desaturase, has a 552 nucleotide-long 5′ UTR intron (intron 1), and the other seven introns are located in the coding sequence. The splicing of the 5′ half region of the NtFAD3 was studied through an in vivo splicing assay using Arabidopsis leaf explants. The low splicing efficiency of intron 2 was much improved when the assay construct harbored intron 1. Deletion of intron 1 and the replacement of intron 1 to the NtFAD3 intron 8 decreased the splicing efficiency of intron 2. The splicing enhancers were redundant and dispersed in the 5′ splice site-proximal, 284-nucleotides region of intron 1. In addition, the interaction among the cis-elements, i.e., the splicing enhancers in the intron 1 and exon 2, were necessary for the efficient splicing of intron 2. The 5′ UTR intron-mediated constitutive splicing was partially inhibited when an SR-like protein, SR45, was deficient. These results indicated a novel function of the 5′ UTR intron, namely an enhancement of the constitutive splicing.

2가 양이온이 Thiamine Pyrophosphate에 의한 Group I Intron Ribozyme의 Splicing 억제에 미치는 영향

안성준,박인국 한국미생물학회 2002 미생물학회지 Vol.38 No.1

2가 양이온($Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$)이 조효소 thiamine pyrophosphate에 의한 T4파지 티민생합성 유전자 (td) 인트론 RNA의 splicing에 미치는 영향을 조사하였다. $Mg^{2+}$를 30 mM까지 증가시켰을 때 splicing 활성은 농도에 비례하여 증가하였다. 그러나 $Mg^{2+}$이 존재하지 않는 상태에서 0.1-4 mM 농도에 걸쳐 $Zn^{2+}$를 사용한 결과 약 20% 정도의 splicing이 일어났다. 이때 대부분의 splicing product는 인트론-엑손2 및 엑손2였고 엑손1-엑손2는 검출되지 않았다. 그리고 4 mM 농도에서는 RNA가 대부분 가수분해되는 현상이 나타났다. $Mn^{2+}$ 이온은 사용한 농도 범위 (0.1-8 mM)에서 $Zn^{2+}$ 보다는 전반적으로 약간 증가된 splicing 활성을 보였으며 8 mM 농도에서도 약 30% 정도의 splicing 활성을 보였다. $Zn^{2+}$ 이온처럼 splicing product는 인트론-엑손2 및 엑손2였고 엑손1-엑손2는 검출되지 않았 다. 반면에 splicing반응에 10 mM $Mg^{2+}$ 를 첨가했을 때 $Zn^{2+}$ 과 $Mn^{2+}$ 이온은 평균 약 35-40% 정도 splicing활성을 촉진시키는 것으로 나타났다. 실험한 2가 양이온 중에서 특히 $Mg^{2+}$ 은 가장 낮은 농도에서 thiamine pyrophosphate 에 의한 억제반응을 극복하는 최고의 활성효과를 나타냈다. 이와 같은 억제 회복 효과는 $Mg^{2+}$에 의한 리보자임의 td intron 구조적 안정성에 기인하는 것으로 추정된다. Effects of divalent cations such as $Mg^{2+}$, $Mn^{2+}$ and $Zn^{2+}$ on the self-splicing inhibition of the T4 phage thymidylate synthase (td) intron by the coenzyme thiamine pyrophosphate have been investigated. The splicing activity increased in proportion to the concentration of $Mg^{2+}$ up to 30 mM. Without $Mg^{2+}$in the splicing reaction the $Zn^{2+}$ ion tested in the range of 0.1-6 mM concentration only produced the splicing activity about 20% that of the normal splicing rate. A majority of the splicing products were I-E2 and E2 but El-E2 ligation product, Cl and Ll were not detected. Similar patterns of splicing products were also observed with $Mn^{2+}$. At 6 mM $Zn^{2+}$the intron RNA was hydrolyzed. $Mn^{2+}$produced a little higher splicing activity than that of $Zn^{2+}$over the range of concentrations used and at 8 mM about 28% splicing activity was observed. In contrast, $Mn^{2+}\;and\;Zn^{2+}$ ions promoted the splicing activity about 35-40% on an average in the presence of 10 mM $Mg^{2+}$. Of all divalent cations tested, $Mg^{2+}$exhibited the maximum activation effect to counteract the splicing inhibition by thiamine pyrophosphate. This appears to be due to the stabilizing effect of td intron ribozyme structure essential for the catalytic function by $Mg^{2+}$.

Splicing inhibition of U2AF⁶⁵ leads to alternative exon skipping

Cho, Sunghee,Moon, Heegyum,Loh, Tiing Jen,Jang, Ha Na,Liu, Yongchao,Zhou, Jianhua,Ohn, Takbum,Zheng, Xuexiu,Shen, Haihong National Academy of Sciences 2015 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.112 No.32

<P><B>Significance</B></P><P>Transcription is a biological procedure in which DNA is transcribed to an RNA molecule. However, only fragments of this RNA are needed for protein synthesis. These fragments are exons that are interrupted by introns. Introns are removed by so-called RNA splicing process. Some exons could be alternatively included or excluded from the final RNA molecule. In this study, we have found that U2 snRNP auxiliary factor 65 kDa (U2AF<SUP>65</SUP>), a general splicing regulator, can significantly promote the exclusion of alternative exons. Strikingly, U2AF<SUP>65</SUP> suppresses flanking intron splicing of alternative exons, and even constitutive intron splicing. We deduce that the stimulatory effects of U2AF<SUP>65</SUP> on alternative exon exclusion are induced by the splicing inhibitory effects of U2AF<SUP>65</SUP>.</P><P>U2 snRNP auxiliary factor 65 kDa (U2AF<SUP>65</SUP>) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF<SUP>65</SUP> stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (<I>SMN</I>) pre-mRNA. A stronger 5′ splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF<SUP>65</SUP>. U2AF<SUP>65</SUP> overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF<SUP>65</SUP> inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF<SUP>65</SUP> effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF<SUP>65</SUP> even inhibits general splicing of adenovirus major late (Ad ML) or β-globin pre-mRNA. Thus, we conclude that U2AF<SUP>65</SUP> possesses a splicing Inhibitory function that leads to alternative exon skipping.</P>

Histone and RNA-binding protein interaction creates crosstalk network for regulation of alternative splicing

Kim, Yong-Eun,Park, Chungoo,Kim, Kyoon Eon,Kim, Kee K. Elsevier 2018 Biochemical and biophysical research communication Vol.499 No.1

<P><B>Abstract</B></P> <P>Alternative splicing is an essential process in eukaryotes, as it increases the complexity of gene expression by generating multiple proteins from a single pre-mRNA. However, information on the regulatory mechanisms for alternative splicing is lacking, because splicing occurs over a short period via the transient interactions of proteins within functional complexes of the spliceosome. Here, we investigated in detail the molecular mechanisms connecting alternative splicing with epigenetic mechanisms. We identified interactions between histone proteins and splicing factors such as Rbfox2, Rbfox3, and splicing factor proline and glutamine rich protein (SFPQ) by <I>in vivo</I> crosslinking and immunoprecipitation. Furthermore, we confirmed that splicing factors were bound to specific modified residues of histone proteins. Additionally, changes in histone methylation due to histone methyltransferase inhibitor treatment notably affected alternative splicing in selected genes. Therefore, we suggested that there may be crosstalk mechanisms connecting histone modifications and RNA-binding proteins that increase the local concentration of RNA-binding proteins in alternative exon loci of nucleosomes by binding specific modified histone proteins, leading to alternative splicing. This crosstalk mechanism may play a major role in epigenetic processes such as histone modification and the regulation of alternative splicing.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Rbfox proteins interact with distinctive histone proteins. </LI> <LI> Modified histone proteins bind directly to RNA-binding proteins. </LI> <LI> Methylation of histone proteins regulates AS. </LI> </UL> </P>

Predicting family-wide consensus sequence of pre-mRNA splicing signals in legumes and their potential utility as phylogenetic markers

Choi, Hong-Kyu,Lee, Chae-Young,Kim, Goon-Bo,Nam, Young-Woo,Schraufnagle, Kenneth R.,Chung, Young-Soo,Lee, Jai-Heon,Kim, Doh-Hoon,Jung, Ho-Won 한국식물생명공학회 2013 Plant biotechnology reports Vol.7 No.2

Recent studies have shown that an intron is not merely "junk", but something that plays important roles in many biological processes such as gene expression regulation and alternative splicing. For purposes of studying intron structures and predicting consensus splice motifs, a total of 102 legume species were used to isolate introns across the family. Of 196 gene-targeted PCR primer pairs, we successfully amplified 118 intron-containing genes (60.2 %) and obtained a total of 1,870 introns with an average size of 143 nucleotides, ranging from 61 to 1,036. Species-based compilation of 5'- and 3'-splicing motifs showed, to some extent, lineage-specific conservation in each splicing motif. Compilation of the entire intron set permitted prediction of the consensus sequences of splicing signal motifs in legumes, $A^YG^W\underline{GT}A^BA^BG^H$ and $T^VNC/T\underline{AG}G^HT^V$ for the 50SS and 30SS, respectively. Interestingly, these consensus motifs are very similar to the corresponding genome-wide splicing signals of two model systems, Arabidopsis and rice. This result suggests conservation of pre-mRNA splicing mechanism occurring in higher plants. Multiple alignments of CALTL introns demonstrated that the BP-3'SS region was relatively more conserved than the 5'SS-BP region. We speculate that length of the BP-3'SS region needs to be retained for the interaction with U2AF protein. Phylogenetic analysis demonstrates that each of three splicing motifs is not only phylogenetically informative, but also relevant to evolutionary divergence of species. This result suggests that the splice signal sequences would be a useful tool for the molecular phylogenetic analysis. We also anticipate that gene-targeted amplification in multiple genomes, described in this study, would facilitate studies on intron-located functional elements involved in gene expression regulations.

New Splicing Variants of the Murine Damaged DNA Binding 2

Jun Hong Park,YoonYi Nam,Jungkee Kwon,Jun-Gyo Suh 한국실험동물학회 2010 Laboratory Animal Research Vol.26 No.1

Damaged DNA binding (DDB) protein is an important gene in the repair of damaged DNA. DDB is a heterodimer (DDB1 and DDB2) protein, murine DDB2 has 10 exons about 1.5kb in size (Genbank Accession No. AY027937). Here we identified five DDB2 variants (M1-M5) from various mouse tissues that are generated by alternative splicing. We used reverse transcription-PCR (RT-PCR) to identify splicing variants and isolated PCR products using an agarose-gel PCR purification kit. All isolated PCR products were cloned and the structure of splicing variants was confirmed by sequencing. The first splicing variant M1 was generated by omission of exon 4. The second splicing variant M2, by omission of exons 4-5. The third variants M3 was generated by omission from the middle of exon 1 to exon 6 and was expressed in the heart. Fourth variants M4 was generated by omission of exon 2 and exons 4-7. M5, the last splicing variant was generated by omission of exons 4-7. M4 and M5 were expressed in the spleen. Analysis of tissue distribution by RT-PCR indicates that M1 is most highly expressed in the mouse brain. These results indicated that murine DDB2 has five splicing variants and splicing variants expression patterns were different depending on mouse tissue. Further functional studies of each splicing variants will provide more information about the molecular mechanism of DDB2 function and DDB2 gene expression regulation.

Tau mis-splicing in the pathogenesis of neurodegenerative disorders

( Sun Ah Park ),( Sang Il Ahn ),( Jean Marc Gallo ) 생화학분자생물학회(구 한국생화학분자생물학회) 2016 BMB Reports Vol.49 No.8

Tau proteins, which stabilize the structure and regulate the dynamics of microtubules, also play important roles in axonal transport and signal transduction. Tau proteins are missorted, aggregated, and found as tau inclusions under many pathological conditions associated with neurodegenerative disorders, which are collectively known as tauopathies. In the adult human brain, tau protein can be expressed in six isoforms due to alternative splicing. The aberrant splicing of tau pre-mRNA has been consistently identified in a variety of tauopathies but is not restricted to these types of disorders as it is also present in patients with non-tau proteinopathies and RNAopathies. Tau mis-splicing results in isoform-specific impairments in normal physiological function and enhanced recruitment of excessive tau isoforms into the pathological process. A variety of factors are involved in the complex set of mechanisms underlying tau mis-splicing, but variation in the cis-element, methylation of the MAPT gene, genetic polymorphisms, the quantity and activity of spliceosomal proteins, and the patency of other RNA-binding proteins, are related to aberrant splicing. Currently, there is a lack of appropriate therapeutic strategies aimed at correcting the tau mis-splicing process in patients with neurodegenerative disorders. Thus, a more comprehensive understanding of the relationship between tau mis-splicing and neurodegenerative disorders will aid in the development of efficient therapeutic strategies for patients with a tauopathy or other, related neurodegenerative disorders. [BMB Reports 2016; 49(8): 405-413]

2P-174 Nanoplasmonic system of RNA folding and assembly while pre-mRNA splicing is done

유병선,( Anh H. Nguyen ),심상준 한국공업화학회 2017 한국공업화학회 연구논문 초록집 Vol.2017 No.1

RNA splicing plays important roles in transcriptome and proteome diversity. Herein, we describe the use of a nanoplasmonic system that unveils RNA folding and assembly during pre-mRNA splicing wherein the quantification of mRNA splice variants is not taken into account. With a couple of SERS-probes and plasmonic probes binding at the boundary sites of exon-2/intron-2 and intron-2/exon-3 of the premature RNA of the β-globin gene, the splicing process brings the probes into the plasmonic bands. For plasmonic probes, a plasmon shift increase of ∼29 nm is measured by plasmonic coupling. The increased scattering intensity and SERS fingerprinting reveal the clear dynamics of premRNA splicing. Moreover, a time-resolved experiment of individual RNA molecules exhibited a successful splicing and an inhibited splicing event by 33 μM biflavonoid isoginkgetin, a general inhibitor of RNA splicing. The results show that the RNA splicing is successfully monitored with the nanoplasmonic system.

Relative strength of 5’ splice-site strength defines functions of SRSF2 and SRSF6 in alternative splicing of Bcl-x pre-mRNA

( Namjeong Choi ),( Yongchao Liu ),( Jagyeong Oh ),( Jiyeon Ha ),( Claudia Ghigna ),( Xuexiu Zheng ),( Haihong Shen ) 생화학분자생물학회(구 한국생화학분자생물학회) 2021 BMB Reports Vol.54 No.3

Bcl-x, a member of the Bcl-2 family, plays a key role in apoptosis. Alternative splicing of Bcl-x pre-mRNA through alternative 5’ splice-site selection produces an anti-apoptotic mRNA isoform that includes exon 2b and a pro-apoptotic Bcl-x mRNA isoform that excludes exon 2b. Here we used Bcl-x minigene and identified SRSF2 and SRSF6 as two regulatory factors of 5’ splice-site selection of Bcl-x pre-mRNA. We selected binding clusters closer to 5’ splice-sites from multiple potential binding sites of SRSF2 and SRSF6 to perform loss of functions analysis through site-directed mutagenesis. Our results demonstrated that these mutations did not abolish regulatory functions of SRSF2 or SRSF6, indicating that a single binding motif or a cluster was not a functional target of these proteins in Bcl-x pre-mRNA splicing. Random deletion mutagenesis did not disrupt the role of SRSF2 and SRSF6. Importantly, mutagenesis of 5’ splice-site to a conserved or a weaker score demonstrated that the weaker strength of the target 5’ splice-site or higher strength of the other 5’ splice-site strength limited the role of SRSF2 and SRSF6 in 5’ splice-site activation. [BMB Reports 2021; 54(3): 176-181]

신경내분비 유전자 조절에 있어서 RNA 스플라이싱의 역할 : 시상하부 Gonadotropin-Releasing Hormone(GnRH) 신경호르몬을 중심으로 Special reference to GnRH neurohormone

한진,김경진 한국뇌학회 2001 한국뇌학회지 Vol.1 No.1

고등 진핵생물에서 일차 전사체의 변조적 스플라이싱 메커니즘은 한 개의 유전자로부터 다양한 전사체를 만들어 낼 수 있다는 점에서 유전자의 용량을 증가시켜 주는 강력한 장치이다. 시상하부에서 합성되는 생식소 자극 호르몬 방출 호르몬 (GnRH)은 고등생물의 생식과 발생에 관련된 신경내분비 네트웍의 중요한 조절자이다. GnRH 유전자는 4개의 exon과 3개의 intron으로 구성되어 있으며, 다양한 신경, 호르몬 자극에 의해서 유전자 발현이 조절된다. GnRH 유전자의 전사체 역시 변조적 스플라이싱 단계를 거쳐 적어도 세 가지의 전사체를 만들어 내게 되는데, 이는 각각, 첫 번째 intron인 intron A를 포함하는 형, 두 번째 exon인 exon2를 포함하지 않은 형, 그리고 정상적으로 스플라이싱이 완성된 형이다. 최근, 본 연구실에서는 GnRH 일차 전사체가 정상적으로 스플라이싱이 일어나기 위해서는 exon에 존재하는 exonic splicing enhancer(ESE)의 도움이 필요하며, 이러한 ESE들은 GnRH 신경세포에 존재하는 신경세포 특이적 스플라이싱 인자와 상호 작용함으로써 완전한 형태의 GnRH 전사체를 만들어 낼 가능성이 있음을 밝혀 내었다. 본 논단에서는 신경내분비 유전자 조절에 있어서 RNA 스플라이싱의 역할에 대해 GnRH 유전자를 중심으로 살펴 보고자 하며, 특히 GnRH 일차 전사체의 스플라이싱에 있어서의 ESE의 역할과 이와 관계하는 trans-acting 인자에 대해서 논의하고자 한다. In higher eukaryotes, alternative splicing of pre-mRNA is a powerful and versatile regulatory mechanism by which a single gene can produce multiple transcripts. Hypothalamic gonadotropin-releasing hormone(GnRH) is a pivotal regulator of neuroendocrine network in the mammalian reproduction and development. GnRH gene consists of 4 exons and 3 introns and its gene expression is regulated by a variety of neural and hormonal signals. GnRH gene pre-mRNA undergoes alternative splicing, producing at least three pre-mRNAs, intron A-retaining species, exon2-skipping species, as well as normally spliced form. Recently we demonstrated that exonic splicing enhancers(ESEs) are required for normal RNA processing of GnRH pre-mRNA. Furthermore, these ESEs appear to interact with a GnRH neuron-specific splicing factor, which is a prerequisite for producing mature GnRH mRNA. In this review we will discuss the role of the ESEs in GnRH premRNA splicing, and the trans-acting splicing factors that are involved in neuron-specific GnRH pre-mRNA splicing.