Najmeh Nikpour and Fiona Alum provided suggestions and edited this manuscript. Department of Biochemistry at McGill University supported HM and VM with the CIHR Training Initiative in Chemical Biology. This work was supported by Canadian Institute of Health Research (CIHR) grant 119464 (to R. S.).

아래 프로토콜은 형광 계 RNA 편집 분석을 수행하기위한 절차를 설명한다. 분석은 단일 PCR 튜브, 실험의 범위에 따라 96 - 웰 또는 384 - 웰 플레이트에서 수행 될 수있다. 이어서 형광 신호는 적합한 실시간 PCR 검출 시스템에서 판독 될 수있다. 여기서 분석은 384 - 웰 플레이트의 맥락에서 설명된다. 1. 배양 T. brucei 세포 <ol><li> T.위한 성장 배지를 준비 brucei 형태의 세포를 procyclic. 매체의 1 L의 경우 : <ol><li> 800 ㎖의 miliQ 물에 25.4 g SDM-79 분말을 용해. </li><li> 10 M NaOH로 7.3 2 탄산 수소 나트륨의 g의 산도를 추가합니다. </li><li> 900 ㎖, 소독 필터의 최종 볼륨에 nanopure 물을 추가합니다. </li><li> 10 %의 최종 농도에 태아 혈청 (FBS), 페니실린 - 스트렙토 마이신 솔루션 헤민 (2.5 ㎎ / ㎖)를 추가 (V / V), 100 U / ㎖ 및 7.5 ㎎ / L이었다. </li></ol></li><li> T. &lt;300 ㎖를 성장EM&gt; 1.5 × 10 7 세포 / ml의 밀도로 70 rpm으로 흔들어 28 ° C에서 brucei의 1.7A 야생형 (procyclic 양식) 세포. 참고 :이과 활성 editosome 3 ㎖를 생산해야 ~ 600 편집 반응에 대한 충분한 총 단백질, 0.5 MG. </li><li> 4 ℃에서 10 분 동안 6,000 XG에서 원심 분리하여 세포를 수확 </li><li> 냉장 PBSG 버퍼 50 ㎖ (10 mM의 나 2 HPO 4, 10 밀리미터의 NaH 2 PO 4, 145 mM의 NaCl을, 6 mM의 포도당)와 펠렛을 씻어 10 분에서 10,000 XG에서 원심 분리하여 다시 세포를 스핀 다운 4 ° C. </li></ol> 원유 미토콘드리아의 2. 격리 참고 : 모든 단계를 editosome 활동을 유지하기 위해 얼음 또는 4 ° C에서 수행해야합니다. <ol><li> DTE 완충액 30 ㎖ (1 mM 트리스 - 염산 pH를 8.0 및 1 mM의 EDTA)에서 수확 한 세포를 재현 탁. 위로 쓰다듬​​어 아래로 얼음에 10 배 이상으로 세포막을 방해하는 40 ㎖ 멸균 다운스 호모 게 나이저 (사전 냉장)를 사용합니다. </리&gt; <li> 우선적으로 미토콘드리아를 가지고, 4 ° C에서 10 분 동안 15,800 XG에 0.25 M. 원심 분리기의 최종 농도에 균질로, 즉시 4.3 ml의 60 % 자당 (즉, 1.75 M W / V)를 추가합니다. </li><li> STM 버퍼 (20 MM 트리스 - 염산 산도 8.0, 250 MM의 자당과 2 mM의 MgCl2를) 4.6 ㎖에 미토콘드리아 펠렛을 Resuspend. I 각각 0.3 ㎜, 9 U / ㎖의 최종 농도 0.1 M 염화칼슘의 13.8 μL 및 RNA 분해 효소가없는 DNA 분해 효소의 4 μl를 추가합니다. 얼음에 1 시간 동안 혼합물을 품어. </li><li> 4 ℃에서 10 분 동안 15,800 XG에서 DNA 분해 효소 I. 원심 분리기를 불 활성화 STE 버퍼의 4.6 ML (20 MM 트리스 - 염산 산도 8.0, 250 MM의 자당과 2 mM의 EDTA)를 추가 </li><li> 400 용해 버퍼의 μL (10 MM 트리스 - 염산의 pH 7.2, 10 mM의 MgCl2를, 100 ㎜의 KCl, 1 ㎍ / ㎖의 펩 스타틴, 1 mM의 DTT 및 1X 완전한 EDTA없는 단백질 분해 효소 억제제) 및로 전송에서 펠렛을 재현 탁 의 미세 관. </li><li> 1 %의 최종 농도를 10 % 트리톤 X-100을 추가차 튜브 회전에서 4 ° C에서 15 분 동안 해물을 품어. </li><li> 4 ° C에서 15 분 동안 17,000 XG에 두 번 원심 분리하여 미토콘드리아 해물을 취소; 취소 뜨는 때마다 유지. </li></ol> 3. Editosome 정화 <ol><li> 부어 10 ㎖의 10 % -30 % (40 mM의 HEPES 산도 7.9, 20 밀리미터 밀리그램 (OAC) 2, 100 mM의 KCl을 2 배 HHE 그라데이션 버퍼를 사용하여 초 원심 분리기 튜브 (V / V) 선형 글리세롤 그라데이션 (표 1), 및 2 mM의 EDTA) 및 설명서를 다음으로 그라데이션 메이커. </li><li> 조심스럽게 글리세롤 그라데이션의 상단에서 솔루션 500 μl를 제거하고 부드럽게 지워 미토콘드리아 용해 액 500 μl를로드합니다. 초 원심 분리기를 사용하여 4 ° C에서 6 시간 동안 178,000 XG에 스핀. </li><li> 4 ° C에서 그라데이션의 위에서 아래로 순차적으로 500 μL의 분수를 수집 그런 다음 사용 때까지 -80 ° C에서 액체 질소와 저장소를 사용하여 분수를 동결 스냅. </li></ol><ol><li> 3 분 90 ° C에서 가열하여 1:1의 몰비 T7 프로모터 올리고 뉴클레오타이드 (5&#39;-TAATACGACTCACTATAGGG-3 &#39;)와 T7 프로모터 서열 (표 2)에 상보 시퀀스를 포함하고, 실온에서 냉각 각각의 DNA 템플릿을 어닐링 적어도 10 분. </li><li> 사용 설명서에 따라 시험 관내 전사 키트를 사용하여 RNA를 고쳐 쓰다. </li><li> 7 M 요소 염료 (7 M 요소, 0.05 % 크실렌 Cynol, 0.05 % 브로 모 페놀 블루)의 동일한 볼륨을 추가하여 전사 반응을 중지합니다. 9 %는 폴리 아크릴 아마이드 겔 (9 % 아크릴 아미드, 7 M 요소, 1X TBE)을 변성 살균 필터를 실행합니다. </li><li> 찾을 수있는 단파 UV 램프와 소비세 각각의 RNA와 자외선 (UV) 그림자를 사용합니다. 미세 원심 분리 튜브에 절개 된 젤 조각을 놓고 겔 용출 버퍼 400 μL (20 MM 트리스 - 염산의 pH 7.5, 250 ㎜의 NaOAc, 1 ㎜ EDTA 0.25 % SDS)를 추가합니다. 튜브 회전기에 RT에서 밤새 용리. </li><li> 프리 시즌차가운 100 % 에탄올 1 ㎖를 추가하고 30 분 또는 -20 ° C 하룻밤 -80 ° C에서 하나를 배양하여 용출 RNA를 pitate. </li><li> RNA를 펠렛 4 ° C에서 30 분 동안 16,000 XG에 원심 분리기. </li><li> 75 %의 에탄올 1 ㎖로 펠렛을 씻으십시오. 4 ℃에서 20 분 동안 16,000 XG에 원심 분리기 </li><li> 표 2에 나타낸 바와 같이, 원하는 농도를 달성하기의 RNase없는 물에 적절히 RNA 펠렛을 재현 탁. </li></ol> 5. 형광 기반의 RNA 편집 분석 <ol><li> 하나의 반응,, 미세 원심 분리 튜브에 preA6Rbz 1 pmol의 (1 μL) 2.5 pmol의 gA6Rbz (1 μL) (1:2.5 몰비)를 결합하여 3 분 동안 70 º C에서 배양 해에 대한 RT에 앉아 보자 적어도 10 분. </li><li> 한편 (25 mM의 HEPES pH가 7.9, 10 mM의 밀리그램 (OAC) 2, 50 mM의 KCl을 10 mM의 EDTA), 1 mM의 1X HHE 버퍼를 포함하는 편집 반응, preA6Rbz 및 gA6Rbz 않고, 표 3을 이용하여 마스터 믹스를 준비ATP, 5 MM의 염화칼슘, Torula 효모 RNA, 0.1 % 트리톤 X-100, 및 정제 editosome 16 NG / μL. </li><li> 마스터 믹스를 완료하기 위해 단련 preA6Rbz 및 gA6Rbz를 추가합니다. </li><li> RNase가없는 물 2 μL (무 화합물과 우물) 또는 200 μM의 화합물의 2 μL 하나를 포함하는 우물에 마스터 믹스 (표 3)의 18 μl를 분배하고 그림 5에 따라 플레이트 제어 샘플이 (가) 있습니다. </li><li> 접시 실러와 플레이트를 밀봉하고 공기 방울을 제거하기 위해, 아래 판을 회전. 4 시간 동안 28 ℃에서 배양한다. </li><li> 물론 각 gA6Rbz 경쟁의 25 pmol의 (2 μL)를 추가합니다. 아래 판을 회전 및 실시간 PCR 기계에 배치, 신선한 봉인을 놓습니다. 다음과 같은 실험을 프로그램 : 1 단계 : 5 분 동안 85 ° C; 2 단계 : 10 분 동안 24 ° C; 3 단계 : 중지합니다. </li><li> 23 μL의 최종 볼륨에 각 웰에 FRET 기판의 15 pmol의 (1 μL)를 추가합니다. 신선한 실러와 플레이트를 밀봉.신속하게 판을 회전 및 실시간 PCR 시스템에 다시 배치합니다. </li><li> 다음 단계로 새로운 실험을 프로그램 : 1 단계 : 1 분 37 ° C; 2 단계 : 읽기; 3 단계 : 1, 40 번 단계로 이동합니다; 4 단계 : 중지합니다. </li><li> 독서를 필요로 FAM 필터를 선택하는 모든 우물을 선택하여 설치 판을. 23 μL 및 실행을 시작으로 입력 볼륨. </li><li> 분석 용 막대 그래프에 슬로프를 플로팅하여 운동 측정을 얻기 위해 각 웰 / 샘플에서 얻은 값의 기울기를 계산한다. NOTE : 운동 판독은 시료와 배경 사이의 신호 대 잡음비를 향상시킨다; 배경 샘플은 제로에 가까운 경사를하는 것처럼. 엔드 포인트 독서는 높은 배경 잡음이 있습니다. </li></ol>

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Searching+for+a+wrench+to+throw+into+the+splicing+machine.">Searching for a wrench to throw into the splicing machine.</a> Nature chemical biology. 4, 3-6 (2008).</li><li>Spahn, C., Prescott, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Throwing+a+spanner+in+the+works:+antibiotics+and+the+translation+apparatus.">Throwing a spanner in the works: antibiotics and the translation apparatus.</a> Journal of molecular medicine. 74, 423-439 (1996).</li></ol>

The authors have nothing to disclose.

Trypanosomes의 RNA 편집 복잡한에 대한 억제제를 식별하는 새로운 높은 처리량 검사 방법은 trypanosomatids으로 인한 질병에 대응하기 위해 약물 발견을위한 새로운 도구를 제공하고, 제시했다. FRET 기반 리보 자임 분석은 광범위하게 다양한 용도 20-22에 사용되었다; 그러나, 우리는 RNA 편집 활동 (19)의 생체 모니터링을위한 FRET 기반 리보 자임의 분석의 능력을 활용했다. 이 분석은...

RNA 편집, mRNA의 전사 후 개질 공정은, 제 1 trypanosomatids에서 발견되었다. 그 이후로, 실질적인 작업은 Trypanosoma brucei 2,3에서 RNA 편집의 뒤에 기계를 연구 실시하고있다. 일련의 효소 반응에서 editosome 약 20 단백질의 코어 복합체는, 에너지 발생 산화 적 인산화 시스템의 여러 구성 요소를위한 성숙한 미토콘드리아의 mRNA를 생성한다. 촉매 이벤트의 순서는 가이드 RNA를 (gRNAs)에 의해 결정으로, endonucleolytic 분열, 우리 딜 (U) 추가 또는 삭제, 결찰입니다 4. 코어 editosome 복잡한 단백질 외에도 소품 요인도 5-7을 확인되었다. 이 단백질은 주로 독립적 인 단지로 분류 볼 수 있습니다. 그러나, 코어 editosome 복잡 단백질 어셈블리의 순서와 소품과 코어 복합체의 상호 작용 패턴착물은 아직 결정되지하다. trypanosomatids에서 RNA 편집 과정을 대상으로하는 화학 dissectors를 제공 할 수 있다는 editosome 복합체의 조립 및 기능을 공부에 도움. 또한, 여러 editosome 단백질의 기능 연구는 약 8 ~ 12을 대상으로 자신의 잠재력을 나타내는 다른 생활 단계에 걸쳐 요점을 보여 주었다. 따라서 editosome의 발견 억제제도 trypanosomatids 대하여 리드 화합물로서 작용할 수있다. trypanosomatid으로 인한 질병에 대한 현재 사용 가능한 약물, 독성이 비효율적이고 비용이 13, 14로이시의 적절하다. 효율적이고 편리한 시험 관내 분석은 RNA 편집을 차단 특정 억제제 화학 우주를 탐구 할 필요가있다. 세 가지 분석법이 개발 editosome 활동을 모니터링하는 데 사용되었다 : 시험 관내 RNA 편집 세이 15 (a) 전체 라운드, (b) 시험 관내 RNA 편집 분석 16,17에 미리 절단ND (C) 망치 리보 자임 (HHR) 분석 18 기반. 처음 두 개의 분석 실험은 방사능의 도움으로 편집 된 제품 (ATPase의 6의 mRNA)의 직접 시각화에 의존하고 있습니다. HHR 기반 분석은 편집시 리보 자임로 동작하는 모델로 ATPase의 6의 mRNA의 수정 된 버전을 사용합니다. 기능 리보 자임은 특히 기자로서, 방사성 동위 원소 표지 된 RNA 기판을 절단한다. 최근 Moshiri는 외. 개발 &#39;혼합 측정&#39;방사성 표지 된 RNA 기판은 형광 공명 에너지 전달 (FRET) 기판 (19)으로 교체되는 경우 RNA 편집을 모니터하기 위해 시험 관내 리포터 분석에서 HHR 기반한다. 이 분석의 원리 장점이 있습니다 : (A)는 활성 리보 자임 및 기판 절단의 생산이 낮은 볼륨 (즉, 20 μL)에서 동일한 관에서 동시에 발생하는, 신속하고 편리한 믹스와 분석의 측정 유형입니다, (B )는 방사성 표지 된 물질의 사용을 피하고, (c) 감도 즉이다형광 마이크로 타이 터 플레이트 포맷으로 계측하고, (d) 노이즈 비율이 높은 신호에 의해 fforded. 이 분석을 이용하여, 정제 된 editosome 대하여 공지 RNA 편집 리가 아제 억제제의 효과 (19)를 확인 하였다. 이 실험은 주로 T.에서 전체 editosomes에 대해, RNA 편집 억제제의 신속한 식별을 위해 분석을 확인 brucei. 도 1의 상세한 단계별 개략적 인 형광 계 관내 RNA 편집 분석에서. 이 프로토콜은 두 시험 관내에서 RNA 편집을 모니터링하거나 쉽게 다양한 스케일의 화합물 라이브러리를 스크리닝 적응 될 위해 사용될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="979">
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			<td height="21" style="height:21px;width:391px;">Name of Material/ Equipment</td>
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			<td height="20" style="height:20px;">SDM-79 Medium</td>
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			<td height="23" style="height:23px;">Fetal Bovine Serum</td>
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			<td>heat inactivation at 550C for 1 h</td>
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			<td style="width:139px;">H5533-10G</td>
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			<td height="21" style="height:21px;width:391px;">Penicillin-Streptomycin Sollution</td>
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			<td height="21" style="height:21px;width:391px;">T7 RiboMax Express Large&#160; scale RNA&#160; production system</td>
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			<td height="21" style="height:21px;width:391px;">Kimble Kontes Dounce Tissue Grinders&#160;&#160;</td>
			<td style="width:203px;">Fisher Scientific</td>
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			<td height="21" style="height:21px;width:391px;">Gradient Master, ver 5.25&#160;</td>
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			<td height="21" style="height:21px;width:391px;">Ultra Clear Tube, 13.2ML</td>
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			<td height="21" style="height:21px;width:391px;">SW 41 Ti ROTOR</td>
			<td style="width:203px;">Beckman Coulter</td>
			<td style="width:139px;">331336</td>
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			<td height="21" style="height:21px;width:391px;">MicroSeal &#39;B&#39; Seal, Seals</td>
			<td style="width:203px;">Biorad</td>
			<td style="width:139px;">MSB1001</td>
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			<td height="21" style="height:21px;width:391px;">CFX 384 Touch Real-Time PCR Detection System</td>
			<td style="width:203px;">Biorad</td>
			<td style="width:139px;">185-5484</td>
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			<td height="21" style="height:21px;width:391px;">Acryl/Bis solution (19: 1), 40% (w/v)</td>
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			<td height="21" style="height:21px;width:391px;">Urea, Molecular biology grade, 1Kg</td>
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</table>

rna catalyst as a reporter for screening drugs against rna editing in trypanosomes

실질적인 진전은 trypanosomes에있는 미토콘드리아 RNA 편집의 메커니즘을 결정을했다. 마찬가지로, 상당한 진전은 RNA 편집을 촉매 editosome 착체의 구성 요소를 식별하는 제되었다. 그러나, 그 단백질이 함께 작동하는 방법을 아직 분명하지 않다. editosome 대하여 높은 처리량 스크린에서 얻어진 화합물은 편집주기에서 하나 이상의 단계를 차단하거나 영향을 미칠 수있다. 따라서, 새로운 화합물의 확인은 editosome 기능과 어셈블리를 해부에 대한 가치있는 분자 프로브를 생성합니다. 이전 연구에서, 시험 관내 편집 분석법은 무선 - 표지 된 RNA를 사용하여 수행 하였다. 이러한 분석은 시간에 비효율적이고 높은 처리량의 목적에 적합하지, 소모적이다. 여기서, 균일 한 형광 기반의 &quot;혼합 측정&quot;RNA 편집을 모니터링하는 망치 리보 자임 체외 기자의 분석을 제시한다. 만의 RNA 편집의 결과로해머 헤드 리보 자임 형광 공명 에너지 전달 (FRET) 올리고 리보 뉴클레오타이드 기판 분열을 거친다. 이것은 차례로하여 신호를 생성하는 형광 소광로부터의 분리를 초래한다. editosome 기능이 저해되는 경우 반대로, 형광 신호가 급냉 될 것이다. 이 체외 RNA 편집 또는 editosome 기능을 억제 할 수있는 화학 물질의 높은 처리량 검사에 모니터에 일반적으로 적용되어야 매우 민감하고 간단한 분석이다.

대형 화면을 설정하기 위해 필요한 필수 단계를 설명하기 위해 2-5 분석의 질과 관련된 대표적인 제어 실험이다 피규어. 이러한 검사의 며칠 동안 일관된 분석 또는 다른 화면의 비교를위한 필수 제어 실험이다. 형광 신호 대 잡음비를 평가할 대규모 설정에서 형광 표지 된 올리고 리보 뉴클레오타이드 기판의 안정?...

Watch this Scientific Journal Video about RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes at JoVE.com

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

Trypanosomes에서 RNA 편집에 대한 마약 검사에 대한 기자로 RNA 촉매

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

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8.4K 조회수.  McGill University. A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

비디오: RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

머릿속 RNA 현지화에서 Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

연구

생물학

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HELA 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA 정제에 의한 염색질 절연 (처프)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / 카스 시스템의 Endonucleases을위한 기판 생성

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

모기 내장 Metagenomic RNA-seq에 대한 Ribosomal RNA의 고갈

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; 현장에서</em전체 마운트 태아 및 세포 조직학에서&gt; 하이브리드는 해양 Elasmobranchs을위한 적응

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Zebrafish의 유충 루시 페라 제 리포터 시스템과 글루코 코르티코이드 신호에 대한 화학적 스크리닝 절차

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA 결합 단백질의 검출에 의해<em&gt; 체외</em지방 세포 문화&gt; RNA 풀다운

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

RNA 방해의 실용적인 사용: Liposome 캐리어 바퀴벌레에 대 한 이중 가닥 RNA의 구두 납품

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

이미징 FITC-dextran 규제 Exocytosis 위한 기자로 서

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...