Conclusion

Adding extra nucleotides which were not encoded in the genome gives cells a chance to control the fate of transcripts. It has been shown that one uridine addition to pre-miRNA is capable of modulating its processing (Heo et al., 2012). In this study, I investigated two types of nontemplated nucleotide addition, uridylation and adenylation in human cells and Drosophila, respectively. With the techniques based on high-throughput sequencing, I sought to examine how widespread the modifications take place, which enzyme catalyzes them, and what the functional consequences are.

In this thesis work, I found that TUT4 and TUT7 redundantly uridylate deadenylated mRNAs, and that the uridylated mRNAs are targeted by multiple decay factors for rapid degradation. Because U-tail may serve as a general decay signal, it would be important to uridylate only authentic targets which must be eliminated. I suggest the molecular mecha- nism how U-tails are strictly found on short poly(A) tails (<~25 nt); first, two enzymes have an intrinsic property to act preferentially on short poly(A) tails and second, PABP cooperatively inhibits uridylation of long poly(A) tails (>25 nt) as a fail safe mechanism.

By doing so, uridylation leads deadenylated mRNAs to decay pathways as a fast track. I expect that the current model of uridylation-dependent decay that I propose here would be applied to biological contexts where rapid clearance of mRNA is required.

Next, I improved TAIL-seq to increase sensitivity for poly(A) ⁺ mRNAs in order

to apply to minute amounts of samples (<5 ug). Using highly sensitive mTAIL-seq, I

discovered that cytoplasmic polyadenylation mainly occurs in late oogenesis of Drosophila

and is largely carried out by Wispy, a noncanonical PAP. Furthermore, I addressed the

biological impact of cytoplasmic polyadenylation during Drosophila late oogenesis and

egg activation. I was able to profile and define a group of genes that gets polyadenylated,

which contributes to translational activation depending on the length of poly(A) tail. In

the future, it will be interesting to find the mechanism how specific transcripts are subject

to cytoplasmic polyadenylation and translation.

In conclusion, I developed TAIL-seq and mTAIL-seq based on high-throughput

sequencing and unveiled the characteristics of two distinct post-transcriptional modifica-

tions. Deep sequencing allowed me to investigate mRNA tails at transcriptome-scale with

high-resolution, which was infeasible with traditional biochemical methods. I expect that

the observations made in this study will expand into the research on other types of RNA

tailing, which would ultimately inform us a better understanding of post-transcriptional

modifications.

국문초록

진핵생물의 전령 RNA 꼬리에 관한 전사체 수준의 연구

진핵생물의전령RNA는안정성과번역능력에중대한영향을미치는수많은전사후 변형과정을겪는다.새로합성된전령RNA는

5′

말단에

7-메틸구아닌

모자와

3′

말단 에긴아데닌꼬리를얻는다. 그런정규적인변형이외에도,최근연구들은 유리딘화, 구아닌화같은비주형핵산추가, N6-메틸아데노신,슈도유리딘화와같은리보핵산의 염기변형

,

아데닌

-

이노신교정등다양한종류의변형을전령

RNA

의전사체후표식으 로밝혀내었다.

RNA

꼬리를유전체전체수준에서연구하기위해,나는긴아데닌꼬리길이와

3′

말단변형을정밀하게측정하는꼬리서열분석법(TAIL-seq)을개발하였다.신기하게 도,나는포유동물세포의긴아데닌꼬리길이가평균적으로

50–100 nt

이며,그끝에 광범위한유리딘화와구아닌화가일어난다는것을발견했다.그리고유리딘꼬리는주 로짧은아데닌꼬리(<~25 nt)에존재한것으로보아전령RNA의분해에관여할것으로 생각되었다.

유리딘화는 다양한종의전령RNA에서발견되어왔는데,그기작과중요성은아직 알려지지않았다

.

나는최근개발한꼬리서열분석법을적용해서

TUT4

와

TUT7(TUT4/7,

또는

ZCCHC11/6)효소가

포유류의전령RNA유리딘화를담당한다는것을알아내었다.

유리딘화는짧은아데닌꼬리를가지는전령RNA에일어나는데,시험관에서정제된

TUT4/7도

선택적으로짧은아데닌꼬리를인지하고유리딘꼬리를붙이는것이관찰

되었다.게다가, PABPC1은긴아데닌꼬리를가지는전령RNA의 유리딘화를억제해서 짧은아데닌꼬리에만유리딘화가일어나도록도와주었다.이두개의효소를실험적 으로제거한세포에서는전령RNA의 유리딘꼬리가사라지면서분해가느려지는것이 확인되었고

,

전령

RNA

의분해를담당하는 단백질을억제하면유리딘꼬리가축척되었 다.이와일치하게,마이크로RNA는그표적의 유리딘화를촉진하며,표적의효과적인

분해를위해

TUT4/7이

필요하다는것을발견되었다. 나는이번연구를통해짧은아

데닌꼬리에선택적인 유리딘화가일어나는기작을밝히고,전령RNA분해의 일반적인

표지로서유리딘화의중요성을제시한다.

두번째로,나는이전버전보다전령RNA를약

1,000배

효율적으로서열분석할수 있는새로운버전의꼬리서열분석법

(전령RNA

꼬리서열분석법또는

m꼬리서열분석

법)을보고한다. 기존꼬리서열분석법은다양한종류의

3′

말단정보를줄수있지만, 전령RNA에대한민감도는떨어져서적은양의생물학적시료에는적용하기어려웠다.

나는개선된방법으로초파리난자와배아에서긴아데닌꼬리가조절되는현상을최 초로연구했다.나는모체로부터전달된전령RNA의아데닌화가대부분수정 전,후기 난자발생과정중에일어나며,난자활성화이후더조절되는것을발견했다. 또한이

과정에서

Wispy

효소가리보좀단백질을만드는

RNA를

제외한대부분의전령RNA를

아데닐화시킨다는것을알아내었다.나는이번데이터를 리보좀프로필분석자료와 비교해서난자활성화과정중에 아데닌꼬리의길이와번역효율사이에강한상관관계 가생긴다는것을발견하였다.이번연구는난자에서일어나는긴아데닌꼬리조절이 이후배아의번역환경을형성하는데중요하며

,

동물발생의시작을결정한다는점을 제시한다.높은민감도,적은비용,기술적견고함,넓은적용성등의 장점으로, m꼬리 서열분석법은앞으로전령RNA꼬리를연구하는데유용한도구가될것으로기대한다.

종합적으로

,

나는이번연구에서내가 개발한서열분석방법인꼬리서열분석법

과

m꼬리서열분석법을 이용해,

진핵생물의전령RNA꼬리를연구하였고,그것이전

령RNA안정성과번역에미치는역할을보고하였다.

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