The differential transcriptional activity of two amphibian U1 small-nuclear RNA genes correlates with structural...

We previously analyzed the transcription of an axolotl U1 small-nuclear RNA (snRNA) gene (AmU1) by microinjection into Xenopus laevis oocytes. In such an assay, AmU1 showed a low template activity compared to that of an X. laevis U1 snRNA gene (XlU1B2). Swapping the proximal sequence element (PSE) with that of XlU1B2 was required for AmU1 to acquire a transcription level equal to that of XlU1B2. In the present work, we examine the functional importance of the nucleotides that are common or different in both PSEs with the aim of identifying which nucleotides within the Xenopus U1 PSE are critical for this enhancement of Ambystoma mexicanum U1 snRNA transcription. The PSE mutation analysis showed that the central, phylogenetically conserved C-58/C-57 doublet is absolutely required for U1 promoter activity. In the 3' portion of this element, a CGC to ATG change (positions -54/-52) which partially restores the XlU1B2 PSE sequence, enables the AmU1 gene to gain the same transcriptional activity as XlU1B2. Remarkably, in this clustered point mutation, the sole C-54 to A-54 change is sufficient to obtain this increased level. Therefore, the activity of the AmU1 gene in injected Xenopus oocytes is strongly affected by a single sequence difference between AmU1 and XlU1B2 PSEs. This finding underscores the crucial importance of the nucleotide identity at position -54 to the function of the Xenopus U1 PSE.