Intermitochondrial cement (IMC) harbors piRNA biogenesis machinery and exonuclease domain-containing proteins EXD1 and EXD2 in mouse spermatocytes.

BACKGROUND: Germ granules are large cytoplasmic ribonucleoprotein complexes that emerge in the germline to participate in RNA regulation. The two most prominent germ granules are the intermitochondrial cement (IMC) in meiotic spermatocytes and the chromatoid body (CB) in haploid round spermatids, both functionally linked to the PIWI-interacting RNA (piRNA) pathway. AIMS: In this study, we clarified the IMC function by identifying proteins that form complexes with a well-known IMC protein PIWIL2/MILI in the mouse testis. RESULTS: The PIWIL2 interactome included several proteins with known functions in piRNA biogenesis. We further characterized the expression and localization of two of the identified proteins, Exonuclease 3'-5' domain-containing proteins EXD1 and EXD2, and confirmed their localization to the IMC. We showed that EXD2 interacts with PIWIL2, and that the mutation of Exd2 exonuclease domain in mice induces misregulation of piRNA levels originating from specific pachytene piRNA clusters, but does not disrupt male fertility. CONCLUSION: Altogether, this study highlights the central role of the IMC as a platform for piRNA biogenesis, and suggests that EXD1 and EXD2 function in the IMC-mediated RNA regulation in postnatal male germ cells.

Lysine methylation mapping of crenarchaeal DNA-directed RNA polymerases by collision-induced and electron-transfer dissociation mass spectrometry.

Enzymatic machineries fundamental for information processing (e.g., transcription, replication, translation) in Archaea are simplified versions of their eukaryotic counterparts. This is clearly noticeable in the conservation of sequence and structure of corresponding enzymes (see for example the archaeal DNA-directed RNA polymerase (RNAP)). In Eukarya, post-translational modifications (PTMs) often serve as functional regulatory factors for various enzymes and complexes. Among the various PTMs, methylation and acetylation have been recently attracting most attention. Nevertheless, little is known about such PTMs in Archaea, and cross-methodological studies are scarce. We examined methylation and N-terminal acetylation of endogenously purified crenarchaeal RNA polymerase from Sulfolobus shibatae (Ssh) and Sulfolobus acidocaldarius (Sac). In-gel and in-solution protein digestion methods were combined with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) mass spectrometry analysis. Overall, 20 and 26 methyl-lysines for S. shibatae and S. acidocaldarius were identified, respectively. Furthermore, two N-terminal acetylation sites for each of these organisms were assessed. As a result, we generated a high-confidence data set for the mapping of methylation and acetylation sites in both Sulfolobus species, allowing comparisons with the data previously obtained for RNAP from Sulfolobus solfataricus (Sso). We confirmed that all observed methyl-lysines are on the surface of the RNAP.

Archaeal transcription: making up for lost time.

In recent years, emerging structural information on the aRNAP (archaeal RNA polymerase) apparatus has shown its strong evolutionary relationship with the eukaryotic counterpart, RNA Pol (polymerase) II. A novel atomic model of SshRNAP (Sulfolobus shibatae RNAP) in complex with dsDNA (double-stranded DNA) constitutes a new piece of information helping the understanding of the mechanisms for DNA stabilization at the position downstream of the catalytic site during transcription. In Archaea, in contrast with Eukarya, downstream DNA stabilization is universally mediated by the jaw domain and, in some species, by the additional presence of the Rpo13 subunit. Biochemical and biophysical data, combined with X-ray structures of apo- and DNA-bound aRNAP, have demonstrated the capability of the Rpo13 C-terminus to bind in a sequence-independent manner to downstream DNA. In the present review, we discuss the recent findings on the aRNAP and focus on the mechanisms by which the RNAP stabilizes the bound DNA during transcription.

Soaking of DNA into crystals of archaeal RNA polymerase achieved by desalting in droplets.

Transcription is a fundamental process across the three domains of life and is carried out by multi-subunit enzymatic DNA-directed RNA polymerases (RNAPs). The interaction of RNAP with nucleic acids is tightly controlled for precise and processive RNA synthesis. Whilst a wealth of structural information has been gathered on the eukaryotic Pol II in complex with DNA/RNA, no information exists on its ancestral counterpart archaeal RNAP. Thus, in order to extend knowledge of the archaeal transcriptional apparatus, crystallization of Sulfolobus shibatae RNAP (molecular mass of ~400 kDa) with DNA fragments was pursued. To achieve this goal, crystal growth was first optimized using a nanoseeding technique. An ad hoc soaking protocol was then put into place, which consisted of gently exchanging the high-salt buffer used for apo-RNAP crystal growth into a low-salt buffer necessary for DNA binding to RNAP. Of the various crystals screened, one diffracted to 4.3 Å resolution and structural analysis showed the presence of bound DNA [Wojtas et al. (2012). Nucleic Acids Res. 40, doi:10.1093/nar/gks692].

Structural and functional analyses of the interaction of archaeal RNA polymerase with DNA.

Multi-subunit RNA polymerases (RNAPs) in all three domains of life share a common ancestry. The composition of the archaeal RNAP (aRNAP) is not identical between phyla and species, with subunits Rpo8 and Rpo13 found in restricted subsets of archaea. While Rpo8 has an ortholog, Rpb8, in the nuclear eukaryal RNAPs, Rpo13 lacks clear eukaryal orthologs. Here, we report crystal structures of the DNA-bound and free form of the aRNAP from Sulfolobus shibatae. Together with biochemical and biophysical analyses, these data show that Rpo13 C-terminus binds non-specifically to double-stranded DNA. These interactions map on our RNAP-DNA binary complex on the downstream DNA at the far end of the DNA entry channel. Our findings thus support Rpo13 as a RNAP-DNA stabilization factor, a role reminiscent of eukaryotic general transcriptional factors. The data further yield insight into the mechanisms and evolution of RNAP-DNA interaction.