• BMB Reports
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• 제42권4호
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• pp.185-188
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• 2009

Transcripts contain introns that are usually removed from premessenger RNA (MRNA) in the process of pre-mRNA splicing. After splicing, the mature RNA is exported from the nucleus to the cytoplasm. The splicing and export processes are coupled. UAP56 protein, which is ubiquitously present in organisms from yeasts to humans, is a DExD/H-box family RNA helicase that is an essential splicing factor with various functions in the prespliceosome assembly and mature spliceosome assembly. Collective evidence indicates that UAP56 has an essential role in mRNA nuclear export. This mini-review summarizes recent evidence for the role of UAP56 in pre-mRNA splicing and nuclear export.

• Molecules and Cells
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• 제37권1호
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• pp.1-8
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• 2014

Mammalian-cell messenger RNAs (mRNAs) are generated in the nucleus from precursor RNAs (pre-mRNAs, which often contain one or more introns) that are complexed with an array of incompletely inventoried proteins. During their biogenesis, pre-mRNAs and their derivative mRNAs are subject to extensive cis-modifications. These modifications promote the binding of distinct polypeptides that mediate a diverse array of functions needed for mRNA metabolism, including nuclear export, inspection by the nonsense-mediated mRNA decay (NMD) quality-control machinery, and synthesis of the encoded protein product. Ribonucleoprotein complex (RNP) remodeling through the loss and gain of protein constituents before and after pre-mRNA splicing, during mRNA export, and within the cytoplasm facilitates NMD, ensuring integrity of the transcriptome. Here we review the mRNP rearrangements that culminate in detection and elimination of faulty transcripts by mammalian-cell NMD.

• Animal cells and systems
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• 제1권1호
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• pp.87-91
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• 1997

Effects of divalent cations such as $Mg^{2+}$, $Mn^{2+}$, $Ca^{2+}$, and $Zn^2$ on splicing activity of phage T4 thymidylate synthase intron RNA have been investigated. At the concentration of 0.5 mM, $Mn^{2+}$ in the absence of $Mg^{2+}$, a very small amount of pre-RNA was cleaved into ligation products (El-E2) but no circular or linear intron was produced. As the concentration of $Mn^{2+}$ was increased from 1 to 5 mM the pre-RNA was completely hydrolyzed. In the presence of 5 mM $Mg^{2+}$, both the linear intron and circular intron were produced but no El-E2 ligation product was produced. At both 3 and 5 mM $Mn^{2+}$ the RNA was hydrolyzed completely as observed with no $Mg^2+$ being present. In the case of $Zn^{2+}$, even at 0.5 mM concentration, the pre-RNA was completely hydrolyzed. This observation suggested that $Zn^{2+}$ facilitates RNA hydrolysis more rapidly than $Mn^{2+}$ does. at 5mM $Ca^{2+}$, the RNA was not hydrolyzed and remained intact as a primary transcript.

• Molecules and Cells
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• 제27권6호
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• pp.657-665
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• 2009

The ovomucoid pre-mRNA has been folded into mini-hairpins adaptable for the RNA recognition motif (RRM) protein binding. The number of mini-hairpins were 372 for pre-mRNA and 83-86 for mature mRNA. The spatial arrangements are, in average, 16 nucleotides per mini-hairpin which includes 7 nt in the stem, 5.6 nt in the loop and 3.7 nt in the inter-hairpin spacer. The constitutive splicing system of ovomucoid-pre-mRNA is characterized by preferred order of intron removal of 5/6 > 7/4 > 2/1 > 3. The 5' splice sites (5'SS), branch point sequences (BPS) and 3' splice sites (3'SS) were identified and free energies involved have been estimated in 7 splice sites. Thermodynamic barriers for splice sites from the least (|lowest| -Kcal) were 5, 4, 7, 6, 2, 1, and 3; i.e., -18.7 Kcal, -20.2 Kcal, -21.0 Kcal, -24.0 Kcal, - 25.4 Kcal, -26.4 Kcal and -28.2 Kcal respectively. These are parallel to the kinetic data of splicing order reported in the literature. As a result, the preferred order of intron removals can be described by a consideration of free energy changes involved in the spliceosomal assembly pathway. This finding is consistent with the validity of hnRNP formation mechanisms in previous reports.

• 생명과학회지
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• 제23권11호
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• pp.1311-1316
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• 2013

hnRNP L은 pre-mRNA에 결합하는 단백질들 중에서 핵심이 되는 단백질이다. hnRNP L은 양이 아주 많은 핵 단백질로서 핵과 세포질을 왕복하는 특성을 지니고 있다. 이 단백질은 염색질 변형(chromatin modification), pre-mRNA 스플라이싱, 인트론이 없는 유전자들에서 유래한 mRNA들의 세포질로의 반출(export), IRES-매개성 번역, mRNA의 안정성 조절, 정자형성과정 등, 세포 내의 여러 가지 과정에 관여하고 있는 것으로 알려져 있다. 이 논문에서는 hnRNP L과 결합하는 세포 내 단백질을 찾아내기 위하여 사람의 간세포 cDNA library를 사용하여 이스트 two-hybrid 탐색 실험을 수행하였다. 그 결과 사람의 간세포에서 IGFBP-4가 hnRNP L과 상호결합하는 새로운 파트너라는 것을 발견하였다. 본 연구를 통하여 hnRNP L이 이스트 two-hybrid 시스템에서 IGFBP-4와 특이적으로 상호 결합한다는 것을 처음으로 발견하였다. 본 연구에서는 또한 이스트 two-hybrid 시스템에서 hnRNP L이 IGFBP-4와 상호결합한다는 점을 in vitro pull-down 실험을 통하여 재확인하였다.

• 한국정보과학회:학술대회논문집
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• 한국정보과학회 2011년도 한국컴퓨터종합학술대회논문집 Vol.38 No.1(A)
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• pp.37-40
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• 2011

선택 스플라이싱 (alternative splicing)은 mRNA (messenger RNA)의 전구체인 pre-mRNA가 mRNA로 전사될 때 pre-mRNA의 엑손 영역들 (exons)이 여러 가지 유형 (pattern)으로 다시 연결되는 과정을 말한다. 선택 스플라이싱에 의해 하나의 유전자로부터 서로 다른 mRNA가 만들어 지고 서로 다른 이소형의 단백질 (protein isoforms)이 생성된다. 현재까지 알려진 선택 스플라이싱의 유형은 약 7가지 종류가 있으며, 유전자의 돌연변이 및 질병과 밀접한 연관성을 가지고 있는 것으로 알려져 있다. 본 연구에서는 차세대 시퀀싱 (Next Generation Sequencing : NGS) 기술로 생성된 RNA-Seq 데이터로부터 각 유전자 영역에 대한 선택 스플라이싱 유형을 분류/추출하는 새로운 알고리즘을 제안한다. 제안된 알고리즘에서는 RNA-Seq 데이터를 DNA 시퀀스와 mRNA 트랜스크립트 시퀀스에 동시 매핑하고, 각 엑손 영역에 정렬된 RNA-Seq 데이터의 커버리지 정보 및 엑손의 접합 (junction) 정보를 이용하여 발현된 트랜스크립트 (transcript)의 종류와 양을 측정한다. 알고리즘의 유효성을 보이기 위하여 시뮬레이션 데이터를 이용한 인간 유전자 영역에서의 선택 스플라이싱 유형 추출 실험을 수행하였으며, 검증된 선택 스플라이싱 DB와 비교, 검증하였다.

• Bulletin of the Korean Chemical Society
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• 제26권12호
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• pp.2033-2037
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• 2005

In vitro selection was used to isolate $Mg^{2+}$-dependent self-cleaving ribozymes with cis-cleavage activity from a pre-tRNA library having 40-mer random sequences attached to 5'-end of E. coli $tRNA^{Phe}$. After 8 rounds of SELEX (Systematic Evolution of Ligands by Exponential Enrichment), RNA molecules which can self-cleave at the high concentration of $Mg^{2+}$ were isolated. The selected ribozymes can carry out the self-cleavage reaction in the presence of 100 mM $Mg^{2+}$ but not in 10 mM $Mg^{2+}$. The cleavage sites of the ribozymes are located at +3 and +4 of $tRNA^{Phe}$, compared with +1 position of 5'-end cleavage site of pre-tRNA by RNase P. New RNA constructs deprived of its D stem-loop, anticodon stem-loop, variable loop and T stem-loop, respectively showed the cleavage specificity identical to a ribozyme having the intact tRNA structure. Also, the new ribozyme fused with both a ribozyme and $tRNA^{Leu}$ showed the cleavage activities at the various sites within its sequences, different from two sites of position +3 and +4 observed in the ribozyme with $tRNA^{Phe}$. Our results suggest that the selected ribozyme is not structural-specific for tRNA.

• BMB Reports
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• 제54권3호
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• pp.176-181
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• 2021

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.

• Clinical and Experimental Reproductive Medicine
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• 제40권1호
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• pp.12-22
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• 2013

Objective: We investigated the norepinephrine transporter (NET) expression in normal and pre-eclamptic placentas and analyzed the invasion activity of trophoblastic cells based on norepinephrine (NE)-NET regulation. Methods: NET and NE expression levels were examined by western blot and enzyme-linked immunosorbent assay, respectively. Trophoblast invasion activity, depending on NE-NET regulation, was determined by NET-small interfering RNA (siRNA) and NET transfection into the human extravillous trophoblast cells with or without NE treatment and invasion rates were analyzed by zymography and an invasion assay. Results: NET mRNA was expressed at a low level in pre-eclamptic placentas compared with normal placentas and NE concentration in maternal plasma increased significantly in pre-eclamptic women compared to normal pregnant women (p<0.05). NET gene upregulation and NE treatment stimulated trophoblast cell invasion up to 2.5-fold (p<0.05) by stimulating matrix metalloproteinase-9 activity via the phosphoinositol-3-kinase/AKT signaling pathway, whereas NET-siRNA with NE treatment reduced invasion rates. Conclusion: NET expression is reduced by inadequate regulation of NE levels during placental development. This suggests that a complementary balance between NET and NE regulates trophoblast cell invasion activities during placental development.

• 대한화학회지
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• 제43권3호
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• pp.307-314
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• 1999

대장균의 RNase P의 RNA 성분인 M1 RNA는 대장균 rnpB 유전자의 주요한 일차전사물인 선구-M1 RNA로부터 3'가공으로 생성된다. 이 가공 활성을 가지고 있는 효소 분획을 부분 정제하고 그 특성을 조사하였다. 이 활성 분획을 높은 염농도에 노출시키면 가공 활성이 불활성화하는 것으로 보아, 가공효소는 여러 효소로 이루어진 효소 복합체인 것으로 추정된다. 이 효소 분획은 화학적 핵산 가수분해효소인 납(II) 이온으로 처리하면 효소 활성을 잃지만, 효소 분획 자체에서 추출한 RNA를 가하면 효소 활성을 되찾는다. 이 결과는 효소 활성에는 RNA 분자가 필요하다는 것을 시사한다. 부분 정제한 효소로 형성되는 절단자리들의 분석 결과도, 3'가공과정이 여러 효소에 의하여 일어나고, 적어도 두 가지 다른 경로로 일어난다는 것을 암시한다.