The development of new biochemical, genetic and imaging techniques has allowed the study of large molecular factories as never before — a point vividly illustrated by Utz Fischer and colleagues in the November issue of Nature Cell Biology. By developing a cell-free reconstitution system for spliceosomal small nuclear ribonucleoproteins (snRNPs), they have started to unravel the events involved in assembly of the spliceosome.

The spliceosome is a large, macromolecular complex required for the folding of pre-messenger RNA into the proper conformation for the removal of introns. It contains four snRNPs, consisting of an snRNA component — U1, U2, U4/U6 or U5 — as well as a set of proteins. Some of these proteins, known as the 'Sm' proteins, are common to all of the snRNPs, and in vitro studies have shown that they assemble in a specific order onto the U snRNAs, without the need for ATP, to form the so-called Sm core.

Four years ago, Fischer and colleagues showed that antibodies against two proteins — the survival of motor neuron (SMN) protein and Gemin2 (also known as SIP1) — could interfere with assembly of the Sm core, indicating that additional factors might be involved in vivo. To test this, the authors have now developed a new in vitro experimental system that closely reproduces the conditions found in vivo.

Fischer and colleagues incubated in-vitro transcribed 32P-labelled U1 snRNA from Xenopus laevis with egg extract, then analysed the formation of complexes by native gel electrophoresis. Using this system they demonstrated that, contrary to previous studies, assembly of the Sm core is an active process that requires the hydrolysis of ATP. They then showed that the Sm proteins cannot assemble on the U1 snRNA if the extract is immunodepleted of SMN or Gemin2.

Surprisingly, addition of SMN and Gemin2 to the immunodepleted extracts could not restore the assembly process. The authors wondered whether other essential factors might also have been immunodepleted, and purified an SMN complex from HeLa cells. They found 16 proteins in this complex, including SMN, Gemin2, the Sm proteins and a number of additional proteins. Addition of this SMN complex to the immunodepleted extracts was able to restore assembly of the Sm core.

This is, say the authors, “the first direct evidence for an essential role for the SMN complex in the formation of the Sm core of U1 snRNP”. And, as SMN has been shown to interact with components of small nucleolar ribonucleoproteins (snoRNPs), it is possible that SMN might act as a general assembly factor for different classes of RNPs. The authors now intend to use their cell-free system to dissect the assembly pathway of the other spliceosomal snRNPs, and to take a closer look at how the SMN complex might be involved in the biogenesis of snoRNPs.