Post transcriptional gene silencing (PTGS) is a recently discovered phenomenon 1. The components of PTGS are being cloned and experiment combined with sequence analysis is helping to elucidate its mechanisms. Study of PTGS is providing links between diverse biological processes such as defence against viruses, RNA metabolism 23 and development 4. The gene for the PTGS impaired Arabidopsis mutant sgs3 was recently cloned 5. An initial analysis of the protein did not reveal any motifs, domains or similarity to any other protein. To help shed light on the function of SGS3 a more detailed analysis of the protein has been carried out.'

After initial PSI-BLAST searches with the sequence of SGS3, weak matches were found to a number of plant proteins. Reciprocal matches can often verify the significance of weak matches. Using residues 85 to 225 of a weakly matching Sorghum bicolor protein (SWISSPROT accession O48878) as a PSI-BLAST 6 query at the NCBI site, using an inclusion E-value of 0.002, SGS3 was found in the second round with an E-value of 0.001. This search also found a number of other plant proteins including the rice gene X product (also known as gene X1) 7.

I have termed the main region of similarity the XS domain after 'rice gene

X

S

After initial alignment of the protein sequences the DNA sequence for each was inspected for possible frameshift errors and incorrect splicing boundaries using tblastn and genewise.

The XS domain containing proteins are predicted by ncoils 9 to contain coiled-coils, which suggests that they will oligomerise. Most coiled-coil proteins form either a dimeric or a trimeric structure. It is possible that different members of the XS domain family could oligomerise via their coiled-coils forming a variety of complexes.

Analysis of the C-terminal region after the central coiled-coil region of rice gene X identifies a second region of conservation termed the XH domain, for 'rice gene

X

A global alignment of the full length of the sequences with an XS domain using the T-Coffee alignment program 10, suggested that many of the proteins contained an N-terminal cysteine/histidine cluster. An alignment of the N-terminal cluster is shown in figure 3. This pattern of conservation suggests a zinc binding domain. Although SGS3 is included in the alignment and conserves the putative zinc ligands, there is no statistical support for its inclusion with standard methods. However given the conservation pattern and presence of the shared XS domain and coiled-coil it seems likely that this is an evolutionarily conserved domain. The rice gene X homologues conserve several lysines within the putative zinc binding domain that suggest it may be a nucleic acid binding domain. An RNA binding function would seem plausible if this larger family of proteins were involved in PTGS as the SGS3 protein appears to be.

Can we infer the function of SGS3 based on its similarity to other XS domain proteins? Unfortunately the members of this family are functionally uncharacterised. However, rice gene X was predicted by Chen et al. to be a transcription factor based on two pieces of evidence: (i) the presence of the coiled-coil as found in other transcription factors such as GCN4 11, (ii) the rice gene X contains a ring zinc finger 7. However, many coiled-coils are found in non-transcription factor proteins weakening the first argument. Ring fingers are now thought to mediate the protein interactions of ubiquitin ligases rather than interact with DNA 12. The Rice gene X is the only member of this family that contains a ring finger. This second piece of evidence no longer points to a transcription factor function, but potentially to a role in ubiquitination. Therefore the evidence used to infer these protein are transcription factors is weak and the inference unlikely to be correct.

In summary this analysis suggests that SGS3 may have a nucleic acid binding function and that a large family of plant proteins containing the novel XS and XH domains may be uncharacterised components in PTGS.