The methylosome, a 20S complex containing JBP1 and pICln, produces dimethylarginine-modified Sm proteins.

snRNPs, integral components of the pre-mRNA splicing machinery, consist of seven Sm proteins which assemble in the cytoplasm as a ring structure on the snRNAs U1, U2, U4, and U5. The survival motor neuron (SMN) protein, the spinal muscular atrophy disease gene product, is crucial for snRNP core particle assembly in vivo. SMN binds preferentially and directly to the symmetrical dimethylarginine (sDMA)-modified arginine- and glycine-rich (RG-rich) domains of SmD1 and SmD3. We found that the unmodified, but not the sDMA-modified, RG domains of SmD1 and SmD3 associate with a 20S methyltransferase complex, termed the methylosome, that contains the methyltransferase JBP1 and a JBP1-interacting protein, pICln. JBP1 binds SmD1 and SmD3 via their RG domains, while pICln binds the Sm domains. JBP1 produces sDMAs in the RG domain-containing Sm proteins. We further demonstrate the existence of a 6S complex that contains pICln, SmD1, and SmD3 but not JBP1. SmD3 from the methylosome, but not that from the 6S complex, can be transferred to the SMN complex in vitro. Together with previous results, these data indicate that methylation of Sm proteins by the methylosome directs Sm proteins to the SMN complex for assembly into snRNP core particles and suggest that the methylosome can regulate snRNP assembly.

SMN, the product of the spinal muscular atrophy gene, binds preferentially to dimethylarginine-containing protein targets.

The survival of motor neurons protein (SMN), the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, functions as an assembly factor for snRNPs and likely other RNPs. SMN binds the arginine- and glycine-rich (RG) domains of the snRNP proteins SmD1 and SmD3. Specific arginines in these domains are modified to dimethylarginines, a common modification of unknown function. We show that SMN binds preferentially to the dimethylarginine-modified RG domains of SmD1 and SmD3. The binding of other SMN-interacting proteins is also strongly enhanced by methylation. Thus, methylation of arginines is a novel mechanism to promote specific protein-protein interactions and appears to be key to generating high-affinity SMN substrates. It is reasonable to expect that protein hypomethylation may contribute to the severity of SMA.

Specific RNA binding by a single C2H2 zinc finger.

Zinc finger proteins with high affinity for human immunodeficiency virus Rev responsive element stem loop IIB (RRE-IIB) were previously isolated from a phage display zinc finger library. Zinc fingers from one of these proteins, RR1, were expressed individually and assayed for RRE-IIB affinity. The C-terminal zinc finger retained much of the binding affinity of the two-finger parent and was disrupted by mutations predicted to narrow the RRE-IIB major groove and which disrupt Rev binding. In contrast, the N-terminal zinc finger has a calculated affinity at least 1000-fold lower. Despite the high affinity and specificity of RR1 for RRE-IIB, binding affinity for a 234-nucleotide human immunodeficiency virus Rev responsive element (RRE234) was significantly lower. Therefore, zinc finger proteins that bind specifically to RRE234 were constructed using an in vitro selection and recombination approach. These zinc fingers bound RRE234 with subnanomolar dissociation constants and bound the isolated RRE-IIB stem loop with an affinity 2 orders of magnitude lower but similar to the affinity of an arginine-rich peptide derived from Rev. These data show that single C2H2 zinc fingers can bind RNA specifically and suggest that their binding to stem loop IIB is similar to that of Rev peptide. However, binding to RRE234 is either different from stem loop IIB binding or the tertiary structure of stem loop IIB is changed within the Rev responsive element.

Specific sequences of the Sm and Sm-like (Lsm) proteins mediate their interaction with the spinal muscular atrophy disease gene product (SMN).

The spinal muscular atrophy disease gene product (SMN) is crucial for small nuclear ribonuclear protein (snRNP) biogenesis in the cytoplasm and plays a role in pre-mRNA splicing in the nucleus. SMN oligomers interact avidly with the snRNP core proteins SmB, -D1, and -D3. We have delineated the specific sequences in the Sm proteins that mediate their interaction with SMN. We show that unique carboxyl-terminal arginine- and glycine-rich domains comprising the last 29 amino acids of SmD1 and the last 32 amino acids of SmD3 are necessary and sufficient for SMN binding. Interestingly, SMN also interacts with at least two of the U6-associated Sm-like (Lsm) proteins, Lsm4 and Lsm6. Furthermore, the carboxyl-terminal arginine- and glycine-rich domain of Lsm4 directly interacts with SMN. This suggests that SMN also functions in the assembly of the U6 snRNP in the nucleus and in the assembly of other Lsm-containing complexes. These findings demonstrate that arginine- and glycine-rich domains are necessary and sufficient for SMN interaction, and they expand further the range of targets of the SMN protein.

Specific RNA binding proteins constructed from zinc fingers.

A zinc finger library with degenerate alpha-helices was displayed on the surface of bacteriophage and proteins that bind human immunodeficiency virus type-1 (HIV-1) Rev response element stem loop IIB (RRE-IIB) RNA or 5S rRNA were isolated. DNA encoding affinity selected zinc fingers was shuffled by recombination in vitro to isolate proteins with higher RNA binding affinity. Proteins constructed in this way bind RNA specifically both in vitro and in vivo. These results demonstrate that RNA substrate specificity of zinc fingers can be changed through mutation of alpha-helices to construct novel RNA binding proteins.

Phage display of RNA binding zinc fingers from transcription factor IIIA.

Zinc fingers in transcription factor IIIA (TFIIIA) contribute differentially to RNA and DNA binding affinity. We investigated whether the same putative alpha-helix amino acids in TFIIIA zinc fingers are essential for both RNA and DNA binding. In published structures, zinc fingers make DNA base contacts through amino acids -1, +2, +3, and +6 of the recognition helix. Alanine substitution at these four positions were made in TFIIIA RNA binding zinc fingers, tz4-7 and DNA binding zinc fingers, tz1-3. Substitution in zinc fingers 4 or 6 of tz4-7 reduced RNA affinity 77- and 38-fold, respectively, whereas substitution in zinc fingers 5 or 7 had little effect. DNA binding affinity of tz1-3 was eliminated by alanine substitution in any one zinc finger. We determined which amino acids supported RNA binding by phage display of a library of zinc finger 4 mutants. Lysine at helix position -1 of zinc finger 4 was conserved in all selected tz4-7 fusions. Point mutation of Lys-1 to alanine in zinc finger 4 reduced tz4-7 RNA affinity 30-fold. We propose that RNA binding by TFIIIA shows similarity to DNA binding in the use of the recognition helix. Helix positions -1 and +2 may have particular significance for RNA binding.

Tyr-129 is important to the peptide ligand affinity and selectivity of human endothelin type A receptor.

Molecular modeling and protein engineering techniques have been used to study residues within G-protein-coupled receptors that are potentially important to ligand binding and selectivity. In this study, Tyr-129 located in transmembrane domain 2 of the human endothelin (ET) type A receptor A (hETA) was targeted on the basis of differences between the hETA and type B receptor (hETB) sequences and the position of the residue on ET receptor models built using the coordinates of bacteriorhodopsin. Replacement of Tyr-129 of hETA by alanine, glutamine, asparagine, histidine, lysine, serine, or phenylalanine results in receptor variants with enhanced ET-3 and sarafotoxin 6C affinities but with unchanged ET-1 and ET-2 affinities. Except for Tyr-129-->Phe hETA, these hETA variants have two to three orders of magnitude lower binding affinity for the ETA-selective antagonist BQ123. Replacement of His-150, the residue in hETB that is analogous in sequence to Tyr-129 of hETA, by either tyrosine or alanine does not affect the affinity of peptide ligands. These results indicate that although transmembrane domain 2 is important in ligand selectivity for hETA, it does not play a significant role in the lack of ligand selectivity shown by hETB. Chimeric receptors have been constructed that further support these conclusions and indicate that at least two hETA regions contribute to ligand selectivity. Additionally, the data support an overlap in the binding site in hETA of agonists ET-3 and sarafotoxin 6C with that of the antagonist BQ123.

The validity of the Carlson Psychological Survey with adolescents.

The present study examined the factorial and predictive validity of the Carlson Psychological Survey with male adolescent offenders (n = 350). Factor analysis revealed that, in general, the Carlson is well represented by five scales. Of the four content scales, Antisocial Tendencies proved to be the best of a variety of predictors of institutional adjustment. Thought Disturbance and Self-Depreciation were unrelated to staff ratings on the Behavior Problem Checklist 'Personality Problem' Scale; however, they were positively correlated with the Social Anxiety and Withdrawal scales of the Jesness Inventory. The pattern of these correlations, as well as those between Antisocial Tendencies and the behavioral data, suggest that the Carlson Psychological Survey may be a good indicator of internalizing versus externalizing behavioral and attitudinal styles.