Piwi-RNAs, the Defenders of the Genome


What are Piwi- or piRNAs ?

Here is the answer:

P-element induced wimpy testis (Piwi)-RNAs, or Piwi-interacting RNA, or piRNAs are small RNAs typically 23 to 35 nucleotides long .

.

During the recent 16 years a group of small RNAs has been recognized as a separate group of RNAs called Piwi-interacting RNAs (piRNAs). This novel class of small non-coding RNAs was found to be associated with Piwi proteins of the Argonaute/Piwi family and was reported as Piwi-interacting RNAs (piRNAs) in 2006. A Pubmed search (06/04/2013) revealed that 618 papers have been published about PIWI genes since 1997, starting with a paper by Lin and Spradling in 1997 in which the two researchers report about a novel group of pumilio mutations that affect the asymmetric division of germline stem cells in the Drosophila ovary.

Pumilio is a posterior group gene. The genes in this group are considered essential for the development of the posterior of the fly. The posterior is the end of the fly opposite to its head.

The piwi gene present in Drosophila encodes a protein with a domain common to Argonaute proteins that is necessary for the correct germ cell formation. These Piwi-type proteins (Piwi, Aubergine) are expressed in male and female germ cells in many animals. Piwi proteins and their associated small RNAs are essential for fertility in mammals. The small RNAs that are associated with Piwi proteins are slightly larger than miRNAs and less homogeneous in size when compared to siRNAs and miRNAs. The literature reports that piRNAs are short, 21 to 35 nucleotide long single-stranded RNAs. pRNAs are longer than miRNAs and have 2’-O-methyl-modified 3’ termini. Similar to miRNAs, piRNAs bind to members of the Argonaute protein family. piRNAs guide PIWI proteins which is a specialized subfamily of the Argonaute proteins that are mainly expressed in germ cells.

BSI can help you with your oligonucleotide based research needs. 

Call us at 1-800-227-0627 or move your mouse to “www.biosyn.com“ !

It appears that most of these RNAs are complementary to many different coding regions of retrotransposons but do not depend on the proteins Drosha or Dicer for their production.

Retrotransposons are repeated sequences scattered throughout the Drosophila and mammalian genomes. They are a class of mobile elements that can move via a copy and paste mechanism. Active retrotransposable elements include Long Interspersed Elements (LINEs), Short INterspersed Elements (SINEs) and SVA (SINE/VNTR/Alu) elements. Retrotransposons are responsible for genetic variation and in some cases can cause diseases if mutated. Approximately 0.27% of all human disease mutations can be attributed to retrotransposable elements.

Li et al. in 2013 report that the sequences of piRNAs are more diverse than any other known class of cellular RNAs and that 8.8 million piRNA reads in a deep sequencing library from adult mouse testis comprise 2.7 million different piRNAs. Further, the scientist report that piRNAs map to large blocks of genomic sequence clusters and that the architecture of these clusters suggest that mature piRNAs derive from precursor transcripts via multiple RNA processing steps. The data reported by this research group demonstrated that long RNA polymerase II transcripts are the precursors of mature piRNAs in mammals, and piRNAs are derived from long, continuous RNAs that are subsequently fragmented and processed into piRNAs.

The biogenesis of piRNAs and their roles in transposon silencing and the function of the Piwi-piRNA pathway in epigenetic and post-transcriptional regulation of gene expression has been investigated in recent years and the function of this pathway in somatic cells has been more systematically characterized recently. These findings revealed the Piwi-piRNA pathway as a more general mechanism of gene regulation.

.

The Piwi-piRNA Pathway

Piwi piRNA pathway

The Piwi-piRNA pathway is thought to exhibit multiple regulatory functions of gene expression in diverse organisms. (This model is modified from Peng and Lin 2013).

In the nucleus: The PIWI-piRNA complex can regulate the methylation of heterochromatin protein 1 (HP1), H(istone)3K9 and DNA. This regulation influences the transcription of selected protein-encoding genes, imprinting loci, or transposons.

In the cytoplasm:  The PIWI-piRNA complex can suppress transposons in the nuage, interact with Hsp90-HOP to influence canalization, or interact with translational initiators (eIF) to ihibit polysomes and subsequent protein tranlocation.

.

The Nuage is a term used for Drosophila melanogaster germline granules.

.

The biogenesis of piRNAs and their roles in transposon silencing and the function of the Piwi-piRNA pathway in epigenetic and post-transcriptional regulation of gene expression has been investigated in recent years and the function of this pathway in somatic cells has been more systematically characterized. These findings revealed the Piwi-piRNA pathway as a more general mechanism of gene regulation.

A proposed model of the piRNA pathway

piRNA pathway

Li et al in 2013 proposed this model for the piRNA biogenesis. In this model primary piRNA transcripts are transcribed by RNA polymerase II and contain 5′ caps, exons and introns as well as poly(A) tails. Pachytene gene transcription is controlled by A-MYB, a member of the MYB (myeloblastosis) family of transcription factors. The model proposes that PLD6 (phospholipase D family, member 6)  determines the 5′ end of piRNA intermediates with lengths of > 30 nucleotides. According to the model these intermediates are then loaded into PIWI proteins. A nuclease is thought to trim the 3’end of the piRNA to the length characteristic of the particular bound PIWI protein. The addition a of 2′-O-methyl group to the 3′ end of the mature piRNA prevents further trimming by the 5’adenosylmethionine-dependent methyltransferase Hen1. (Adapted from Li et al 2013).

Hen1 is a methyltransferase that adds a 2′-O-methyl group to the 3′-end of small RNAs to protects the 3′-end of sRNAs from uridylation activity and subsequent degradation [PMID: 20558712]. In bacteria, Hen1 catalyses the same chemical reaction, but forms a Pnkp/Hen1 heterotetramer and is involved in RNA repair .

.

Even though much progress in characterization the role of piRNAs has been made since 1997 more research is needed to find out how piRNA-encoding loci are recognized and regulated by the transcriptional machinery. At present, RNAs associated with Piwi proteins are thought to arise from the cleavage of dsRNA due to transcription of both strands of a retroposon. It is thought that a small RNA that binds to a retroposon transcript will lead to its destruction and protect the genome by limiting the spread of the retroposon. piRNA-encoding loci are transcribed as long, multi-kilobase precursor transcripts that are processed into various piRNAs in most species, such as flies and mammals. The production of piRNA does not require Dicer and are thought to derive from single-stranded RNA. 

A retroposon is a repetitive DNA fragment or sequence that is inserted into chromosomes after it is reverse transcribed from a RNA molecule. Unlike retrotransposons, retroposons do not encode for reverse transcriptase (RT) and are non-autonomous elements with regard to transposition activity.

Two new recent studies have shed more light on the function of piRNAs. Li et al. in 2013 performed high-throughput sequencing of total RNA and processed piRNAs from six developmental stages of mouse testes. The authors mapped these sequence reads to the corresponding genomic loci. The research group investigated the regulatory mechanisms of the burst of piRNA occuring during the pachytene stage of meiosis during sperm development and production (spermatogenesis).

According to their results group P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) can be distinguished from other animal small silencing RNAs by their longer length (typically 23 to 35 nucleotides long), 2’-O-methyl-modified 3’ termini, and their association with PIWI proteins.

The scientists report that mammalian transposon-silencing piRNAs accumulate early in spermatogenesis and pachytene piRNAs are produced later during postnatal spermatogenesis accounting for >95% of all piRNAs in the adult mouse testis. The authors show that the transcription factor A-MYB initiates pachytene piRNA production. A-MYB drives transcription of both pachytene piRNA precursor RNAs and the mRNAs for core piRNA biogenesis factors including MIWI.

Miwi and Mili proteins are mouse homologs of Piwi and contain a C-terminal Piwi domain. Both proteins bind to piRNAs in male germ cells and are essential for spermatogenesis in mice.

Billi et al. performed a meta-analysis of small RNA-sequencing data sets representing male and female C. elegans germline cells. The scientists compared the upstream elements of the piRNA-encoding loci with male-versus female-specific expression.

Billi et al’s. author summary states:

“Across the animal kingdom, Piwi-interacting piRNAs protect genome integrity and promote fertility. While the functions of piRNAs are well-characterized, far less is known about how they are generated and how their expression is regulated. In the Caenorhabditis elegans genome, a conserved sequence motif lies upstream of many piRNA loci and appears to regulate their expression. We combined computational and experimental approaches to investigate the role of this motif in the expression of piRNAs. We discovered that >70% of piRNAs are differentially enriched in male versus female germline, and these male and female piRNAs show different upstream motifs. Using a transgenic system for expressing synthetic piRNAs in vivo, we demonstrate that variation of a single nucleotide within this motif influences piRNA germline enrichment. We further show that the conserved motif is capable of driving piRNA expression in genomic isolation. Accordingly, the genomic distribution of these motifs determines which sequences are expressed as piRNAs in C. elegans. Our results suggest that each C. elegans piRNA represents an independent transcript whose sequence, abundance, and germline enrichment are encoded by a variant upstream motif, defining a novel modality for expression of piRNAs.”

.

High-throughput sequencing together with chromatin immunoprecipitation (ChiP) was used to define the genomic structure of the piRNA-producing genes.  The ChiP technique has now become the method of choice for the study of RNA- or DNA-protein complexes.

.

Additional Notes

Pachytene is the third stage of the prophase of meiosis during which the homologous chromosomes become short and thick and divide into four distinct chromatids.

Prophase is the first stage of mitosis in which the chromatin condenses into double rod-shaped structures called chromosomes in which the chromatin becomes visible, the nuclear membrane breaks down, and the spindle apparatus forms at opposite poles of the cell.

References

Billi AC, Freeberg MA, Day AM, Chun SY, Khivansara V, Kim JK.; A conserved upstream motif orchestrates autonomous, germline-enriched expression of Caenorhabditis elegans piRNAs. PLoS Genet. 2013;9(3):e1003392. doi: 10.1371/journal.pgen.1003392. Epub 2013 Mar 14.

James Darnell; RNA Life’s indispensable molecule.Cold Spring Harbor Laboratory Press, NY 2011. ISBN 978-1-936113-19-4.

Li XZ, Roy CK, Dong X, Bolcun-Filas E, Wang J, Han BW, Xu J, Moore MJ, Schimenti JC, Weng Z, Zamore PD.; An ancient transcription factor initiates the burst of piRNA production during early meiosis in mouse testes. Mol Cell. 2013 Apr 11;50(1):67-81. doi: 10.1016/j.molcel.2013.02.016. Epub 2013 Mar 21.

Li XZ, Roy CK, Moore MJ, Zamore PD. Defining piRNA primary transcripts. Cell Cycle. 2013 May 10;12(11). 1657-1658.

Lin H, Spradling AC. A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. Development. 1997 Jun;124(12):2463-76.

Peng JC, Lin H.; Beyond transposons: the epigenetic and somatic functions of the Piwi-piRNA mechanism. Curr Opin Cell Biol. 2013 Apr;25(2):190-4. doi: 10.1016/j.ceb.2013.01.010. Epub 2013 Mar 4.

.

BSI can help you with your research needs. 

Long Oligos BSI
Synthetic short, long, and ultra long methylated or non-methylated DNA or RNA fragments are now available.

Call us at 1-800-227-0627 or move your mouse to “www.biosyn.com“ !



Categories: Bioinformatics, Cell Development, Cellular Reprogramming, Chromatin, Development, DNA, Epigenetics, Gene Expression, Gene families, Genome, heterochromatin, LincRNA, lncRNA, Long noncoding RNA, miRNA, non-coding RNAs, Nucleoprotein, piRNA, Piwi-RNA, Protein Families, Regulatory RNA, RNA, RNA Editing, RNA silencing, RNA Structure, RNA Synthesis, RNA World, RNA World Hypothesis, RNAi

Tags: , , , , , , , , , ,

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s

%d bloggers like this: