The 3′ termini of mouse Piwi RNAs are 2′-O-methylated

RNA Synthesis BSI

Kirino & Mourelatos and Ohara et al. showed in 2007 that Piwi RNAs (piRNAs) are 2′-O-methylated at the 3′-end

Several thousands of piwi RNA (piRNA) species that are typically 24–32 nucleotide (nt) long have been found in mammals, zebrafish, and Drosophila. Most of these piRNAs appear to be generated from a small number of long single-stranded RNA precursors that are often encoded by repetitive intergenic sequences in the genome. The term piwi or sometimes also called PIWI was originally called ‘P-element induced wimpy testis’ in Drosophila.

Selective targeting of piRNAs through 2’-O-methylated 3’-ends is achieved by their selective recognition by Piwi PAZ (Piwi/Argonaut/Zwille) domains present in Piwi proteins. Piwi proteins target piRNAs that are methylated at the 2’-OH group at their 3’ ends. Recent progress in RNAs research indicates that piRNAs and Piwi proteins are predominantly present in the germline and play a critical role in genomic defense against transposable elements. This defense mechanism is thought to protect the genome against defects that could be induced by transposable elements during gametogenesis and fertilization.

Let us briefly recall what we learned in undergraduate biology. Gametogenesis is the formation or production of gametes which in most multicellular organisms occurs during meiosis. The germline of an individual are the cells that contain genetic material in a cell lineage that is passed down to a child through the gametes before it is modified by somatic recombination or maturation.

A gamete is a cell that fuses with another cell during fertilization (conception) in sexual reproduction. During Meiosis one copy of each homologous chromosome is separated into each new “gamete”. This process reduces the number of sets of chromosomes by half, so that when gametic recombination (fertilization) occurs the ploidy (the number of sets of chromosomes) of the parent cells will be reestablished. To pass down the chromosomal DNA to the next generation damage to the DNA needs to be prevented. Apparently this is the job of piRNAs.

However, the common belief that PIWI proteins have only germline-restricted functions has been challenged recently. Thomson and Lin in 2009 report in their review paper that piwi genes are found to be overexpressed in several human cancers indicating a more expanded role of piRNAs.

Two scientists, Kirino and Mourelatos, working in the US, as well as a group of scientists working in Japan, showed in 2007 that the 3’ termini of mouse piRNAs are 2’-O-methylated. A cartoon that illustrates the general structure of piRNAs is shown below.


Structural features of piRNAs

piRNA structureStructure of piRNAs

How was the structure of the piRNAs determined?

Approach A:


To determine the nature of the modifications present on piRNAs Kirino and Mourelatos (2007) used a classical biochemical approach that included the following steps:

1.    Purification of total piRNAs from testis

2.   Treatment of RNA with calf intestinal phosphatase (CIP) to remove the phosphate groups on the 5- and 3-ends  

3.   Labeling of the 5’-ends of untreated and treated RNAs with  [γ-32P]ATP using T4 polynucleotide kinase (PNK)

4.   Labeling of the 3’-ends of untreated and treated RNAs with  [γ-32P]pCp and T4 ligase.

5.   Result: The CIP treatment markedly improved the efficiency of 5’ end-labeling. This indicated that piRNA have a phosphate at their 5’-terminal end.

6.  Reacting 5’ end-labeled total piRNAS with sodium periodate (NaIO4) followed by β-elimination

7.  Result: The RNAs treated with NaIO4 migrated faster in denaturing polyacrylamide gels (PAGE) in comparsion to untreated RNAs and synthetic control RNA (27 nt long) that contained 2’- and 3’-hydroxyl groups were shortened by one nucleotide. These results indicated that the 3’ terminal end of the piRNAs was modified either on the 2’- or the 3’-hydroxyl group

8.   Analysis of piRNA nucleotides by two dimensional thin layer chromatography (2D-TLC): 5’- or 3’- end labeled total piRNAs, purified and synthetic (control RNA), were digested completely by Nuclease P1 or RNase T2, respectively and the resultant 5’- or 3’-labeled nucleotides were analyzed by 2D-TLC.  

9.   Results: The 3’-end of the piRNAs are methylated at the 2’-OH group.

Approach B:


The second group of researchers with the lead author Ohara (Ohara et al., 2007) used a more modern approach to determine the nature of the modifications present on piRNAs:

1.   RNAs (3 piRNAs and one control miRNA) were analyzed by northern blotting.

2.   The treatment of these RNAs with NaIO4 and β-elimination changed the migration speed of the miRNA.

3.   Result: The piRNAs were not affected by the treatment. The piRNA are modified at the 3’ end. 

4.   RNA fractions were purified by denautering PAGE. 

5.   Eluted piRNAs were digested with RNase T2.

6.   Digest was analyzed by liquid chromatography and mass spectrometry (LC-MS).  

7.   Results: LC-MS analysis revealed that 3’termini of piRNAs were 2’-O-methylated.

Chemistry for specific reagents for 2-O-methylated residues in RNA.


Chemistry of the periodate and β-elimination reaction


Periodate oxydation 3' ribose

Periodate oxydation of cisdiol residues at the terminal 3-ribose.


Chemistry of the alkaline hydrolysis of 2-O-methylated nucleotides

piRNA cleavage

Selective cleavage of nonmethylated ribose residues at alkaline pH. The 2-O-methylated residues are resistant for such cleavage.


Acylation of free 2' OH

Acylation of ribose free 2-OH by SHAPE reagents N-methylisatoic anhydride (NMIA) and 1-methyl-7-nitro-isatoic anhydride.

SHAPE stands for “Selective 2′-Hydroxyl Acylation and Primer Extension”.


Humans contain four Piwi family proteins, Hiwi1, Hiai2, Hiwi3, and Hili. Tian et al. in 2011 reported the structural models of the Hili-PAZ (Piwi/Argonaute/Zwille) domain in the free state and Hiwi1 PAZ domain bound to self-complementary 14-mer RNAs (12-bp + 2-nt overhang) containing 2′-OCH3 and 2′-OH at their 3′ ends. These structures explain the preferential binding of 2′-OCH3 over 2′-OH 3’ends. On the other hand, the more constricted binding pocket for the human Ago1 PAZ domain exhibits a reversed order with the preferential binding of 2′-OH over 2′-OCH3.

The structure for a piRNA including the location of the 2′-O-Me at the 3′ end in the piRNA and the structural models for Hiwi1 PAZ with 2′-OCH3 RNA [PBD code 2O6E] are shown below.

PIWI RNA structure 2

Ball and stick model of  a 2′-O-Me 3′-RNA is illustrated here. The model was generated with PyMol using the atomic coordinates form the structure with PBD code 3O6E deposited by Tian et al. in the protein structure database.

Different views of the Hiwi1 PAZ-RNA complex containing one PAZ domain bound to the overhang-containing strand are illlustrated below.

PIWI PAZ Structure 2

PIWI PAZ Structure BP 1

PIWI PAZ Structure BP 2

PIWI PAZ Structure BP


Isabelle Behm-Ansmant, Mark Helm, and Yuri Motorin; Review Article: Use of Specific Chemical Reagents for Detection of Modified Nucleotides in RNA. Journal of Nucleic Acids. Volume 2011 (2011), Article ID 408053, 17 pages. doi:10.4061/2011/408053.

Yohei Kirino & Zissimos Mourelatos; Mouse Piwi-interacting RNAs are 2′-O-methylated at their 3′ termini.  NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 14 NUMBER 4 APRIL 2007,  pp347- 348. doi:10.1038/nsmb1218.

Tomoya Ohara, Yuriko Sakaguchi, Takeo Suzuki, Hiroki Ueda, Kenjyo Miyauchi & Tsutomu Suzuki; The 3′ termini of mouse Piwi-interacting RNAs are 2′-O-methylated. NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 14 NUMBER 4 APRIL 2007, pp349-350. doi:10.1038/nsmb1220

Tian Y, Simanshu DK, Ma JB, Patel DJ.; Structural basis for piRNA 2′-O-methylated 3′-end recognition by Piwi PAZ (Piwi/Argonaute/Zwille) domains. Proc Natl Acad Sci U S A. 2011 Jan 18;108(3):903-10. doi: 10.1073/pnas.1017762108. Epub 2010 Dec 30.

Travis Thomson and Haifan Lin; The Biogenesis and Function of PIWI Proteins and piRNAs: Progress and Prospect. Annu. Rev. Cell Dev. Biol. 2009. 25:355–76.

Categories: Bioanalysis, Cancer, Cell Development, Epigenetics, Gametogenesis, Germline, piRNA, Piwi Proteins, Piwi-RNA

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