microRNA介绍

miRNA是一类小分子RNA
Recent discoveries of small regulatory RNA molecules have been keeping the research community abuzz. Thus far, two classes of tiny regulatory RNA molecules have been identified.

siRNAs
The first class encompasses the small interfering RNAs (siRNAs), which have an integral role in the phenomenon of RNA interference (RNAi), a form of post-transcriptional gene silencing. In RNAi, dsRNAs introduced into certain organisms or cells are degraded into ~22 nt fragments. These ~22 nt siRNA molecules then bind to the complementary portion of their target mRNA and tag it for degradation. siRNAs are believed to have a role in conferring viral resistance and in preventing transposon hopping.

stRNAs
The second class of regulatory small RNAs have been referred to as small temporal RNAs (stRNAs). Examples of RNAs in this group include ~22 nt lin-4 and let-7 RNAs. These RNA molecules, which have a role in the temporal regulation of Caenorhabditis elegans development, are initially processed from a ~70 nt ssRNA transcript folded into a stem-loop structure. Upon processing, these stRNAs are thought to prevent translation of their target mRNAs by binding to the target's complementary 3' untranslated regions (UTRs). Interestingly, the same RNase enzyme, Dicer, processes both siRNAs and stRNAs.

A New Class of Small RNA Molecules
Now a new chapter is added to this story. In 3 papers published in the 26 October 2001 issue of Science (ref. 1-3), nearly 100 additional small ~22 nt RNA molecules have been identified. These RNA molecules, termed microRNAs (miRNAs), were discovered in Drosophila, C. elegans, and HeLa cells by the Bartel, Tuschl and Ambros labs. Much like lin-4 and let-7, these miRNAs are formed from precursor RNA molecules that fold into a stem-loop secondary structure. The newly discovered ~22 nt miRNAs are believed to play a role in regulation of gene expression. Based on the biochemical analysis of stRNAs and siRNAs, it has been hypothesized that miRNAs might regulate translation (like lin-4 and let-7) or modify mRNA stability (like siRNAs).

Identifying miRNAs
The 3 research teams used both a biochemical and a bioinformatics approach to identify these miRNAs. Both siRNAs and stRNAs, which are processed by Dicer, are ~22 nt, have a 5' phosphate and have a 3' hydroxyl group. The Bartel and Tuschl laboratories used a modified directional cloning strategy to select for molecules with similar characteristics. In short, RNA molecules were fractionated by size, ligated to 3' and 5' adapters, reverse transcribed and then amplified by PCR. The resulting PCR products were subcloned and sequenced. The location and clustering of the miRNA precursors within the genome was then determined by querying genomic sequence databases. This analysis also helped determine whether the miRNAs were degradation products of mRNAs, tRNAs, or rRNAs (4).

The Ambrose lab also took a different approach to searching for miRNAs. This lab used the RNA folding program 'mfold' to determine whether highly conserved C. elegans and C. briggsae intergenic sites contained potential miRNA precursors. Northern blots were then used to confirm whether these miRNAs were actually expressed.

miRNA Characteristics
The 3 research teams were able to identify almost 100 new miRNAs. These were comprised of 14 Drosophila, 19 HeLa cell, and 60 C. elegans miRNAs. Approximately 15% of these miRNAs were conserved (with 1-2 mismatches) across worm, fly, and mammalian genomes (4). According to Lau and colleagues in the Bartel lab, however, all miRNAs seemed to have orthologs in other species. All of the identified miRNAs were located at either the 3'- or the 5'-side of a stem loop within a ~70 nt RNA precursor. Some of these precursors were so tightly clustered that it was suggested they might be synthesized on the same transcript.

The expression pattern of the miRNAs varied. While some C. elegans and Drosophila miRNAs were expressed in all cells and at all developmental stages, others had a more restricted spatial and temporal expression pattern. This suggested that these miRNAs, like lin-4 and let-7, might be involved in post-transcriptional regulation of 'developmental' genes.

Stumbling Blocks to Identification and Elucidating Function
Although nearly 100 miRNAs were identified using biochemical and bioinformatics approaches, none of the teams were able to identify the miRNA targets using an informatics approach. The failure to do so may be because the entire miRNA does not have to be complementary to the target in order for binding to occur. The lin-4 and let-7 miRNAs, for example, bind to their respective target mRNAs through complementary 5' and 3' regions (with the middle part of the miRNA looping out). These findings (or lack there-of) leave open the question: what do these miRNAs do? Are they involved in post-transcriptional gene regulation, and if so, what are their target genes?

The isolated miRNAs may constitute only a subset of those available. According to Lau and colleagues, "many of the identified miRNAs were represented by only a single clone". Therefore they hypothesized that their "sequencing had not reached saturation" and that some miRNAs were not isolated in this procedure.

The Tuschl, Bartel and Ambros labs have begun what promises to be a long line of research into miRNAs. It appears that the "tiny RNA world" may not be so tiny after all.

References

Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001) Identification of novel genes coding for small expressed RNAs. Science 294:853-858.
Lau, N.C., Lim, L.P., Weinstein, E.G., and Bartel, D.P. (2001) An abundant class of tiny RNAs with probable regulatory roles in Caenorhabditis elegans. Science 294:858-862.
Lee, R.C. and Ambrose, V. (2001) An extensive class of small RNAs in Caenorhabditis elegans. Science 294:862-864.
Ruvkun, G. (2001) Glimpses of a tiny RNA world. Science 294:797-799

新技术探索microRNA的功能

基因表达调控决定了细胞内合成哪些细胞、和成量多少，这个过程是由无数不同分子控制的。其中一种天然调控分子就是小干扰RNA(small interfering RNA,siRNA), siRNA选择性破坏其识别的一种蛋白的生产，这个过程称为RNA干扰。这些短链核苷酸与其它细胞蛋白结合形成一个RNA诱导的沉默复合体（RNA-induced silencing complex, RISC），RISC定位于将编码蛋白质的遗传信息丛细胞核转移到细胞质的蛋白质加工厂－－核糖体内的信使RNA上，对信使RNA产生破坏作用。

生物学家已在动植物中发现了数百个另一种天然短链调控RNA，称为microRNA。同siRNA一样，microRNA也是通过类似甚至完全一样的RISC分子影响基因表达的。然而，动物的microRNA是在蛋白质生产的另一个不同阶段靶向信使RNA的。虽然研究人员已经确定了这些microRNA的序列，但对于揭开它们的功能－－即它们分别破坏哪种蛋白以及这些蛋白有什么作用，进展却十分缓慢，尚未找到有效的研究工具。目前只有4种动物的microRNA功能被破译，尽管该领域是一大热点。

为确定siRNAs 和 microRNAs的功能，Gyorgy Hutvagner和他的同事开发出一个由短链2’-O-methyl寡核苷酸组成的系统，这些寡核苷酸序列与靶siRNA 或 microRNA对应，能与之结合并抑制其功能，这样研究人员就可以对RNA干扰背后的机制有空前的了解。Hutvagner和他的同事根据一个叫做let-7的microRNA构建了一个寡核苷酸抑制因子，let-阻断Lin-41蛋白的生产，对于线虫的正常发育时间选择十分重要。导入这种寡核苷酸的线虫出现let-7缺失线虫的典型特点，说明该抑制因子确实阻断了这个的功能。而且，文章作者还用这种寡核苷酸找到证据证明两种早先认为与let-7有关的蛋白，与其干扰活动直接相关。

应用这种技术，科学家可以快速探查数百个microRNA的生物学功能，以及它们的附件RISC蛋白，甚至探查它们所破坏的基因和蛋白。不仅如此，查明RISC生产是否饱和对于利用RNA干扰来揭示序列已知但功能未知的基因的功能具有重要意义；了解所需siRNA的最小量，研究人员就能避免siRNA的急剧及其伴随的任何不需要的细胞活动。

miRNA调节造血干细胞分化

《Science》刊登了华人科学家程长真（音译）和李宁（音译）采用miRNA来调节造血干细胞的分化，为传统的干细胞分化开辟了新的方法和思路。
全文:http://www.sciencemag.org/cgi/reprint/303/5654/83

miRNA-23控制神经元的分化

MicroRNAs (miRNAs) are 21-23 bp RNA molecules that are processed from larger 70-90 bp stem-looped precursors. To date, over 150 miRNAs have been identified in such diverse organisms as worms, fruit flies, humans, and plants (1). The function of most miRNAs is not known. A few miRNAs, however, have been shown to play roles in developmental timing (in worms) and cell proliferation (in fruit flies) by inhibiting the expression of downstream genes (2). Two mechanisms of gene suppression by miRNAs have so far been identified. In one, miRNAs bind to partially complementary regions in the 3' untranslated region (3'UTR) of target transcripts to suppress translation. In the other, the miRNAs bind to perfectly complementary target mRNAs and degrade them via the RNAi pathway.

In the 19 June 2003 issue of Nature, Kawasaki and Taira report on a novel function for a human miRNA. The authors show that miRNA-23 is involved in the differentiation of a human neuronal cell line--a function that is carried out via translational suppression of the Hes1 gene (3). These findings are significant because a function for a human miRNA is identified for the first time.

A Link Between miRNA-23 and Hes1

In order to identify miRNA-23 target genes, Kawasaki and Taira searched the NCBI DNA database for genes with exact matches to human miRNA-23. In their search the best match was found near the termination codon of Hes1 (77% homology)--a basic helix-loop-helix transcriptional repressor that is part of the Notch signaling pathway.

To determine whether the Hes1 mRNA is actually a target for miRNA-23, the authors used NT2 cells as a model system. NT2 cells are embryonic carcinoma cells that differentiate into neuronal cells upon retinoic acid treatment. The authors hypothesized that this differentiation might be dependent on suppression of Hes1 expression by miRNA-23, since it was known that Hes1 up-regulation suppresses neuronal differentiation (4). To confirm their hypothesis, the authors first determined that the levels of HES1 protein (but not Hes1 mRNA) decrease after NT2 cells are treated with retinoic acid. More importantly, they determined that this decrease is accompanied by an increase in miRNA-23 levels. These results suggested that the increase in miRNA-23 levels was causing the reduction in HES1 protein levels in differentiating NT2 cells.

To confirm that miRNA-23 is directly responsible for suppression of HES1 expression, two approaches were taken. In one, synthetic miRNA-23 was introduced into undifferentiated NT2 cells (with their high HES1 levels and low miRNA-23 levels). This resulted in a decrease in expression levels of HES1 protein. In the other, siRNAs targeting miRNA-23 were introduced into differentiated NT2 cells (with their low HES-1 levels and high miRNA-23 levels). This led to a decrease in miRNA-23 levels and an increase in HES-1 levels.

The Role of miRNA-23 in Differentiation of NT2 Cells

The authors then showed that expression of miRNA-23 is responsible for the differentiation of NT2 cells (presumably via a reduction in HES1 protein levels). To this end, the authors made use of two protein markers--SSEA-3, which is only expressed in undifferentiated NT2 cells, and MAP2, which is only expressed in differentiated NT2 cells. Using these markers, the authors showed that addition of siRNAs targeting miRNA-23 prevents the differentiation of NT2 cells upon retinoic acid treatment. This blockage, however, is reversed by addition of synthetic miRNA-23.

Discussion

The results by Kawasaki and Taira suggest that suppression of HES1 expression by miRNA-23 is necessary for the differentiation of NT2 cells. It is interesting to note that in addition to miRNA-23, several worm and fruit fly miRNAs have been shown to play roles in development. It is therefore possible that other mammalian, worm, or fruit fly miRNAs also play roles in various developmental processes. This premise is supported by the differential spatial and temporal pattern of miRNA expression in various organisms.

To date, over 150 miRNAs have been cloned. These miRNAs have been identified based on their size and not their function. One way to uncover the function of miRNAs is to identify their mRNA target(s). This task has proved difficult because 1) many miRNAs may be only partially complementary to target transcripts and 2) lack of complete homology to target genes makes it is difficult to identify them via alignment programs. In this regard, it is interesting to note that database searches were used to identify Hesl as a miRNA-23 target. These results suggest that database searches may lead to the identification of new miRNA targets, thus paving the way for the identification of novel functions of other miRNAs.

References

Pasquinelli AE. (2002). MicroRNAs: deviants no longer. Trends in Genet. 18(4): 171-173.
Ambros V (2003). MicroRNA pathways in flies and worms: growth, death, fat, stress, and timing. Cell 113:673-676.
Kawasaki H and Taira K. (2003). Hes1 is a target of microRNA-23 during retinoic-acid-induced neuronal differentiation of NT2 cells. Nature 423:838-842.
Ishibashi M, Moriyoshi K, Sasai Y, Shiota K, Nakanishi S, and Kageyama R (1994). Persistent expression of helix-loop-helix factor HES-1 prevents mammalian neural differentiation in the central nervous system. EMBO J. 13:1799-1805.